Compositions and related methods for controlling vector-borne diseases

ABSTRACT

Provided herein are methods and compositions useful for human health, e.g., for targeting one or more microorganisms resident in a host insect (e.g., arthropod, e.g., insect, e.g., pathogen vector), the modulation resulting in a decrease in the fitness of the host. The invention features a composition that includes a modulating agent (e.g., phage, peptide, small molecule, antibiotic, or combinations thereof) that can alter the host&#39;s microbiota in a manner that is detrimental to the host. By disrupting microbial levels, microbial activity, microbial metabolism, or microbial diversity, the modulating agent described herein may be used to decrease the fitness of a variety of insects that carry vector-borne pathogens that cause disease in humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/450,032, filed on Jan. 24, 2017, and U.S. Provisional Application No.62/583,925, filed on Nov. 9, 2017, the contents of which are herebyincorporated herein by reference in their entireties.

BACKGROUND

Insects function as vectors for pathogens causing severe human diseasesuch as dengue, trypanosomiases, and malaria. With 174 million diagnosesand 655,000 million deaths in 2011, malaria is considered as one of themost significant diseases worldwide. Thus, there is need in the art formethods and compositions to control insects that carry vector-bornediseases.

SUMMARY OF THE INVENTION

Disclosed herein are compositions and methods for modulating the fitnessof insects for controlling the spread of vector-borne diseases inhumans. The composition includes an agent that alters a level, activity,or metabolism of one or more microorganisms resident in a host, thealteration resulting in a modulation in the host's fitness.

In one aspect, provided herein is a method of decreasing fitness of avector (e.g., insect vector) for a human pathogen, the method includingdelivering an antimicrobial peptide having at least 90% sequenceidentity (e.g., at least 90%, 92%, 94%, 96%, 98%, or 100% sequenceidentity) with one or more of the following: cecropin (SEQ ID NO: 82),melittin, copsin, drosomycin (SEQ ID NO: 93), dermcidin (SEQ ID NO: 81),andropin (SEQ ID NO: 83), moricin (SEQ ID NO: 84), ceratotoxin (SEQ IDNO: 85), abaecin (SEQ ID NO: 86), apidaecin (SEQ ID NO: 87), prophenin(SEQ ID NO: 88), indolicidin (SEQ ID NO: 89), protegrin (SEQ ID NO: 90),tachyplesin (SEQ ID NO: 91), or defensin (SEQ ID NO: 92) to the vector.

In some embodiments, the delivery includes delivering the antimicrobialpeptide to at least one habitat where the vector grows, lives,reproduces, feeds, or infests.

In some embodiments, the antimicrobial peptide may be delivered in aninsect comestible composition for ingestion by the vector.

In some embodiments, the antimicrobial peptide may be formulated as aliquid, a solid, an aerosol, a paste, a gel, or a gas composition.

In some embodiments, the insect may be at least one of a mosquito,midge, louse, sandfly, tick, triatomine bug, tsetse fly, or flea.

In another aspect, provided herein is a composition including anantimicrobial peptide having at least 90% sequence identity (e.g., atleast 90%, 92%, 94%, 96%, 98%, or 100% sequence identity) with one ormore of the following: cecropin (SEQ ID NO: 82), melittin, copsin,drosomycin (SEQ ID NO: 93), dermcidin (SEQ ID NO: 81), andropin (SEQ IDNO: 83), moricin (SEQ ID NO: 84), ceratotoxin (SEQ ID NO: 85), abaecin(SEQ ID NO: 86), apidaecin (SEQ ID NO: 87), prophenin (SEQ ID NO: 88),indolicidin (SEQ ID NO: 89), protegrin (SEQ ID NO: 90), tachyplesin (SEQID NO: 91), or defensin (SEQ ID NO: 92) formulated for targeting amicroorganism in a vector (e.g., an insect vector) for a human pathogen.

In some embodiments of the second aspect, the antimicrobial peptide maybe at a concentration of about 0.1 ng/g to about 100 mg/g (about 0.1ng/g to about 1 ng/g, about 1 ng/g to about 10 ng/g, about 10 ng/g toabout 100 ng/g, about 100 ng/g to about 1000 ng/g, about 1 mg/g to about10 mg/g, about 10 mg/g to about 100 mg/g) or about 0.1 ng/mL to about100 mg/mL (about 0.1 ng/mL to about 1 ng/mL, about 1 ng/mL to about 10ng/mL, about 10 ng/mL to about 100 ng/mL, about 100 ng/mL to about 1000ng/mL, about 1 mg/mL to about 10 mg/mL, about 10 mg/mL to about 100mg/mL) in the composition.

In some embodiments of the second aspect, the antimicrobial peptide mayfurther include a targeting domain.

In some embodiments of the second aspect, the antimicrobial peptide mayfurther include a cell penetrating peptide.

In yet another aspect, the composition includes an agent that alters alevel, activity, or metabolism of one or more microorganisms resident inan insect host, the alteration resulting in a decrease in the insecthost's fitness.

In some embodiments of any of the above compositions, the one or moremicroorganisms may be a bacterium or fungus resident in the host. Insome embodiments, the bacterium resident in the host is at least oneselected from the group consisting of Candidatus spp, Buchenera spp,Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp,Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidusspp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp,Xylella spp, Erwinia spp, Agrobacterium spp, Bacillus spp, Paenibacillusspp, Streptomyces spp, Micrococcus spp, Corynebacterium spp, Acetobacterspp, Cyanobacteria spp, Salmonella spp, Rhodococcus spp, Pseudomonasspp, Lactobacillus spp, Enterococcus spp, Alcaligenes spp, Klebsiellaspp, Paenibacillus spp, Arthrobacter spp, Corynebacterium spp,Brevibacterium spp, Thermus spp, Pseudomonas spp, Clostridium spp, andEscherichia spp. In some embodiments, the fungus resident in the host isat least one selected from the group consisting of Candida,Metschnikowia, Debaromyces, Starmerella, Pichia, Cryptococcus,Pseudozyma, Symbiotaphrina bucneri, Symbiotaphrina Scheffersomycesshehatae, Scheffersomyces stipites, Cryptococcus, Trichosporon,Amylostereum areolatum, Epichloe spp, Pichia pinus, Hansenula capsulate,Daldinia decipien, Ceratocytis spp, Ophiostoma spp, and Attamycesbromatificus. In certain embodiments, the bacteria is a Wolbachia spp.(e.g., in a mosquito host). In certain embodiments, the bacteria is aRickettsia spp. (e.g., in a tick host).

In any of the above compositions, the agent, which hereinafter may alsobe referred to as a modulating agent, may alter the growth, division,viability, metabolism, and/or longevity of the microorganism resident inthe host. In any of the above embodiments, the modulating agent maydecrease the viability of the one or more microorganisms resident in thehost. In some embodiments, the modulating agent increases growth orviability of the one or more microorganisms resident in the host.

In any of the above embodiments, the modulating agent is a phage, apolypeptide, a small molecule, an antibiotic, a bacterium, or anycombination thereof.

In some embodiments, the phage binds a cell surface protein on abacterium resident in the host. In some embodiments, the phage isvirulent to a bacterium resident in the host. In some embodiments, thephage is at least one selected from the group consisting of Myoviridae,Siphoviridae, Podoviridae, Lipothrixviridae, Rudiviridae,Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae,Cystoviridae, Fuselloviridae, Gluboloviridae, Guttaviridae, Inoviridae,Leviviridae, Microviridae, Plasmaviridae, and Tectiviridae.

In some embodiments, the polypeptide is at least one of a bacteriocin,R-type bacteriocin, nodule C-rich peptide, antimicrobial peptide, lysin,or bacteriocyte regulatory peptide.

In some embodiments, the small molecule is a metabolite.

In some embodiments, the antibiotic is a broad-spectrum antibiotic.

In some embodiments, the modulating agent is a naturally occurringbacteria. In some embodiments, the bacteria is at least one selectedfrom the group consisting of Bartonella apis, Parasaccharibacter apium,Frischella perrara, Snodgrassella alvi, Gilliamela apicola,Bifidobacterium spp, and Lactobacillus spp. In some embodiments, thebacterium is at least one selected from the group consisting ofCandidatus spp, Buchenera spp, Blattabacterium spp, Baumania spp,Wigglesworthia spp, Wolbachia spp, Rickettsia spp, Orientia spp, Sodalisspp, Burkholderia spp, Cupriavidus spp, Frankia spp, Snirhizobium spp,Streptococcus spp, Wolinella spp, Xylella spp, Erwinia spp,Agrobacterium spp, Bacillus spp, Paenibacillus spp, Streptomyces spp,Micrococcus spp, Corynebacterium spp, Acetobacter spp, Cyanobacteriaspp, Salmonella spp, Rhodococcus spp, Pseudomonas spp, Lactobacillusspp, Enterococcus spp, Alcaligenes spp, Klebsiella spp, Paenibacillusspp, Arthrobacter spp, Corynebacterium spp, Brevibacterium spp, Thermusspp, Pseudomonas spp, Clostridium spp, and Escherichia spp.

In any of the above compositions, host fitness may be measured bysurvival, reproduction, or metabolism of the host. In any of the aboveembodiments, the modulating agent may modulate the host's fitness byincreasing pesticidal susceptibility of the host (e.g., susceptibilityto a pesticide listed in Table 12). In some embodiments, the modulatingagent modulates the host's fitness by increasing pesticidalsusceptibility of the host. In some embodiments, the pesticidalsusceptibility is bactericidal or fungicidal susceptibility. In someembodiments, the pesticidal susceptibility is insecticidalsusceptibility.

In any of the above compositions, the composition may include aplurality of different modulating agents. In some embodiments, thecomposition includes a modulating agent and a pesticidal agent (e.g., apesticide listed in Table 12). In some embodiments, the pesticidal agentis a bactericidal or fungicidal agent. In some embodiments, thepesticidal agent is an insecticidal agent.

In any of the above compositions, modulating agent may be linked to asecond moiety. In some embodiments, the second moiety is a modulatingagent.

In any of the above compositions, the modulating agent may be linked toa targeting domain. In some embodiments, the targeting domain targetsthe modulating agent to a target site in the host. In some embodiments,the targeting domain targets the modulating agent to the one or moremicroorganisms resident in the host.

In any of the above compositions, the modulating agent may include aninactivating pre- or pro-sequence, thereby forming a precursormodulating agent. In some embodiments, the precursor modulating agent isconverted to an active form in the host.

In any of the above compositions, the modulating agent may include alinker. In some embodiments, the linker is a cleavable linker.

In any of the above compositions, the composition may further include acarrier. In some instances, the carrier may be an agriculturallyacceptable carrier.

In any of the above compositions, the composition may further include ahost bait, a sticky agent, or a combination thereof. In someembodiments, the host bait is a comestible agent and/or achemoattractant.

In any of the above compositions, the composition may be at a doseeffective to modulate host fitness. I

In any of the above compositions, the composition may be formulated fordelivery to a microorganism inhabiting the gut of the host. In any ofthe above compositions, the composition may be formulated for deliveryto a microorganism inhabiting a bacteriocyte of the host and/or the gutof the host.

In some embodiments, the composition may be formulated for delivery to aplant. In some embodiments, the composition may be formulated for use ina host feeding station.

In any of the above compositions, the composition may be formulated as aliquid, a powder, granules, or nanoparticles. In some embodiments, thecomposition is formulated as one selected from the group consisting of aliposome, polymer, bacteria secreting peptide, and syntheticnanocapsule. In some embodiments, the synthetic nanocapsule delivers thecomposition to a target site in the host. In some embodiments, thetarget site is the gut of the host. In some embodiments, the target siteis a bacteriocyte in the host.

In a further aspect, also provided herein are hosts that include any ofthe above compositions. In some embodiments, the host is an insect. Insome embodiments, the insect is a mosquito, midge, louse, sandfly, tick,triatomine bug, tsetse fly, or flea. In certain embodiments, the insectis a mosquito. In certain embodiments, the insect is a tick. In certainembodiments, the insect is a mite. In certain embodiments, the insect isa louse.

Also provided herein is a system for modulating a host's fitnesscomprising a modulating agent that targets a microorganism that isrequired for a host's fitness, wherein the system is effective tomodulate the host's fitness, and wherein the host is an insect. Themodulating agent may include any of the compositions described herein.In some embodiments, the modulating agent is formulated as a powder. Insome embodiments, the modulating agent is formulated as a solvent. Insome embodiments, the modulating agent is formulated as a concentrate.In some embodiments, the modulating agent is formulated as a diluent. Insome embodiments, the modulating agent is prepared for delivery bycombining any of the previous compositions with a carrier.

In yet a further aspect, also provided herein are methods for modulatingthe fitness of an insect using any of the compositions described herein.In one instance, the method of modulating the fitness of an insect hostincludes delivering the composition of any one of the previous claims tothe host, wherein the modulating agent targets the one or moremicroorganisms resident in the host, and thereby modulates the host'sfitness. In another instance, the method of modulating microbialdiversity in an insect host includes delivering the composition of anyone of the previous claims to the host, wherein the modulating agenttargets the one or more microorganisms resident in the host, and therebymodulates microbial diversity in the host.

In some embodiments of any of the above methods, the modulating agentmay alter the levels of the one or more microorganisms resident in thehost. In some embodiments of any of the above methods, the modulatingagent may alter the function of the one or more microorganisms residentin the host. In some embodiments, the one or more microorganisms may bea bacterium and/or fungus. In some embodiments, the one or moremicroorganisms are required for host fitness. In some embodiments, theone or more microorganisms are required for host survival.

In some embodiments of any of the above methods, the delivering step mayinclude providing the modulating agent at a dose and time sufficient toeffect the one or more microorganisms, thereby modulating microbialdiversity in the host. In some embodiments, the delivering step includestopical application of any of the previous compositions to a plant. Insome embodiments, the delivering step includes providing the modulatingagent through a genetically engineered plant. In some embodiments, thedelivering step includes providing the modulating agent to the host as acomestible. In some embodiments, the delivering step includes providinga host carrying the modulating agent. In some embodiments the hostcarrying the modulating agent can transmit the modulating agent to oneor more additional hosts.

In some embodiments of any of the above methods, the composition may beeffective to increase the host's sensitivity to a pesticidal agent(e.g., a pesticide listed in Table 12). In some embodiments, the host isresistant to the pesticidal agent prior to delivery of the modulatingagent. In some embodiments, the pesticidal agent is an allelochemicalagent. In some embodiments, the allelochemical agent is caffeine,soyacystatin N, monoterpenes, diterpene acids, or phenolic compounds. Insome embodiments, the composition is effective to selectively kill thehost. In some embodiments, the composition is effective to decrease hostfitness. In some embodiments, the composition is effective to decreasethe production of essential amino acids and/or vitamins in the host.

In some embodiments of any of the above methods, the host is an insect.In some embodiments, the host is a vector for a human pathogen. In someembodiments, the vector is a. mosquito, midge, louse, sandfly, tick,triatomine bug, tsetse fly, or flea. In certain embodiments, the vectoris a mosquito. In certain embodiments, the vector is a tick. In certainembodiments, the vector is a mite. In certain embodiments, the vector isa louse.

In some embodiments, the human pathogen is a virus, a protozoan, abacterium, a protist, or a nematoda. In some embodiments, the virus isone belonging to the group Togaviridae, Flaviviridae, Bunyaviridae,Rhabdoviridae, or Orbiviridae. In some embodiments, the bacterium is onebelonging to the genus Yersinia, Francisella, Rickettsia, Orientia, orBorrelia. In some embodiments, the protozoan is one belonging to thegenus Plasmodium, Trypanosoma, Leishmania, or Babesia. In someembodiments, the nematode is one belonging to the genus Brugia. In someembodiments, the composition is effective to prevent or decreasetransmission of the pathogen to humans. In some embodiments, thecomposition is effective to prevent or decrease horizontal or verticaltransmission of the pathogen between hosts. In some embodiments, thecomposition is effective to decrease host fitness, host development, orvectorial competence.

In another aspect, also provided herein are screening assays to identifymodulating agent that modulate the fitness of a host. In one instance,the screening assay to identify a modulating agent that modulates thefitness of a host, includes the steps of (a) exposing a microorganismthat can be resident in the host to one or more candidate modulatingagents and (b) identifying a modulating agent that decreases the fitnessof the host.

In some embodiments of the screening assay, the modulating agent is amicroorganism resident in the host. In some embodiments, themicroorganism is a bacterium. In some embodiments, the bacterium, whenresident in the host, decreases host fitness. In some embodiments of thescreening assay, the modulating agent affects anallelochemical-degrading microorganism. In some embodiments, themodulating agent is a phage, an antibiotic, or a test compound. In someembodiments, the antibiotic is timentin or azithromycin.

In some embodiments of the screening assay, the host may be aninvertebrate. In some embodiments, the invertebrate is an insect. Insome embodiments, the insect is a mosquito. In some embodiments, theinsect is a tick. In certain embodiments, the insect is a mite. Incertain embodiments, the insect is a louse.

In any of the above embodiments of the screening assay, host fitness maybe modulated by modulating the host microbiota.

Definitions

As used herein, the term “bacteriocin” refers to a peptide orpolypeptide that possesses anti-microbial properties. Naturallyoccurring bacteriocins are produced by certain prokaryotes and actagainst organisms related to the producer strain, but not against theproducer strain itself. Bacteriocins contemplated herein include, butare not limited to, naturally occurring bacteriocins, such asbacteriocins produced by bacteria, and derivatives thereof, such asengineered bacteriocins, recombinantly expressed bacteriocins, andchemically synthesized bacteriocins. In some instances, the bacteriocinis a functionally active variant of the bacteriocins described herein.In some instances, the variant of the bacteriocin has at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity, e.g., over a specified region or over the entire sequence, toa sequence of a bacteriocin described herein or a naturally occurringbacteriocin.

As used herein, the term “bacteriocyte” refers to a specialized cellfound in certain insects where intracellular bacteria reside withsymbiotic bacterial properties.

As used herein, the term “effective amount” refers to an amount of amodulating agent (e.g., a phage, lysin, bacteriocin, small molecule, orantibiotic) or composition including said agent sufficient to effect therecited result, e.g., to decrease or reduce the fitness of a hostorganism (e.g., insect, e.g., mosquito, tick, mite, louse); to reach atarget level (e.g., a predetermined or threshold level) of a modulatingagent concentration inside a target host; to reach a target level (e.g.,a predetermined or threshold level) of a modulating agent concentrationinside a target host gut; to reach a target level (e.g., a predeterminedor threshold level) of a modulating agent concentration inside a targethost bacteriocyte; to modulate the level, or an activity, of one or moremicroorganism (e.g., endosymbiont) in the target host.

As used herein, the term “fitness” refers to the ability of a hostorganism to survive, and/or to produce surviving offspring. Fitness ofan organism may be measured by one or more parameters, including, butnot limited to, life span, reproductive rate, mobility, body weight, andmetabolic rate. Fitness may additionally be measured based on measuresof activity (e.g., biting animals or humans) or disease transmission(e.g., vector-vector transmission or vector-human transmission).

As used herein, the term “gut” refers to any portion of a host's gut,including, the foregut, midgut, or hindgut of the host.

As used herein, the term “host” refers to an organism (e.g., insect,e.g., mosquito, louse, mite, or tick) carrying resident microorganisms(e.g., endogenous microorganisms, endosymbiotic microorganisms (e.g.,primary or secondary endosymbionts), commensal organisms, and/orpathogenic microorganisms).

As used herein “decreasing host fitness” or “decreasing host fitness”refers to any disruption to host physiology, or any activity carried outby said host, as a consequence of administration of a modulating agent,including, but not limited to, any one or more of the following desiredeffects: (1) decreasing a population of a host by about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (2) decreasing thereproductive rate of a host (e.g., insect, e.g., mosquito, tick, mite,louse) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%,100% or more; (3) decreasing the mobility of a host (e.g., insect, e.g.,mosquito, tick, mite, louse) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 99%, 100% or more; (4) decreasing the body weight of ahost (e.g., insect, e.g., mosquito, tick, mite, louse) by about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (5)increasing the metabolic rate or activity of a host (e.g., insect, e.g.,mosquito, tick, mite, louse) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 99%, 100% or more; (6) decreasing vector-vector pathogentransmission (e.g., vertical or horizontal transmission of a pathogenfrom one insect to another) by a host (e.g., insect, e.g., mosquito,tick, mite, louse) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99%, 100% or more; (7) decreasing vector-human pathogentransmission (e.g., insect, e.g., mosquito, tick, mite, louse) by about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (8)decreasing host (e.g., insect, e.g., mosquito, tick, mite, louse)lifespan by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%,100% or more; (9) increasing host (e.g., insect, e.g., mosquito, tick,mite, louse) susceptibility to pesticides (e.g., insecticides) by about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; or(10) decreasing vector competence by a host (e.g., insect, e.g.,mosquito, tick, mite, louse) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 99%, 100% or more. A decrease in host fitness can bedetermined in comparison to a host organism to which the modulatingagent has not been administered.

The term “insect” includes any organism belonging to the phylumArthropoda and to the class Insecta or the class Arachnida, in any stageof development, i.e., immature and adult insects.

As used herein, “lysin” also known as endolysin, autolysin, mureinhydrolase, peptidoglycan hydrolase, or cell wall hydrolase refers to ahydrolytic enzyme that can lyse a bacterium by cleaving peptidoglycan inthe cell wall of the bacterium. Lysins contemplated herein include, butare not limited to, naturally occurring lysins, such as lysins producedby phages, lysins produced by bacteria, and derivatives thereof, such asengineered lysins, recombinantly expressed lysins, and chemicallysynthesized lysins. A functionally active variant of the bacteriocin mayhave at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified region orover the entire sequence, to a sequence of a synthetic, recombinant, ornaturally derived bacteriocin, including any described herein.

As used herein, the term “microorganism” refers to bacteria or fungi.Microorganisms may refer to microorganisms resident in a host organism(e.g., endogenous microorganisms, endosymbiotic microorganisms (e.g.,primary or secondary endosymbionts)) or microorganisms exogenous to thehost, including those that may act as modulating agents. As used herein,the term “target microorganism” refers to a microorganism that isresident in the host and impacted by a modulating agent, either directlyor indirectly.

As used herein, the term “agent” or “modulating agent” refers to anagent that is capable of altering the levels and/or functioning ofmicroorganisms resident in a host organism (e.g., insect, e.g.,mosquito, tick, mite, louse), and thereby modulate (e.g., decrease) thefitness of the host organism (e.g., insect, e.g., mosquito, tick, mite,louse).

As used herein, the term “pesticide” or “pesticidal agent” refers to asubstance that can be used in the control of agricultural,environmental, or domestic/household pests, such as insects, fungi,bacteria, or viruses. The term “pesticide” is understood to encompassnaturally occurring or synthetic insecticides (larvicides, andadulticides), insect growth regulators, acaricides (miticides),nematicides, ectoparasiticides, bactericides, fungicides, or herbicides(substance which can be used in agriculture to control or modify plantgrowth). Further examples of pesticides or pesticidal agents are listedin Table 12. In some instances, the pesticide is an allelochemical. Asused herein, “allelochemical” or “allelochemical agent” is a substanceproduced by an organism that can effect a physiological function (e.g.,the germination, growth, survival, or reproduction) of another organism(e.g., a host insect, e.g., mosquito).

As used herein, the term “peptide,” “protein,” or “polypeptide”encompasses any chain of naturally or non-naturally occurring aminoacids (either D- or L-amino acids), regardless of length (e.g., at least2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100, or moreamino acids), the presence or absence of post-translationalmodifications (e.g., glycosylation or phosphorylation), or the presenceof, e.g., one or more non-amino acyl groups (for example, sugar, lipid,etc.) covalently linked to the peptide, and includes, for example,natural proteins, synthetic, or recombinant polypeptides and peptides,hybrid molecules, peptoids, or peptidomimetics.

As used herein, “percent identity” between two sequences is determinedby the BLAST 2.0 algorithm, which is described in Altschul et al.,(1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analysesis publicly available through the National Center for BiotechnologyInformation.

As used herein, the term “bacteriophage” or “phage” refers to a virusthat infects and replicates in bacteria. Bacteriophages replicate withinbacteria following the injection of their genome into the cytoplasm anddo so using either a lytic cycle, which results in bacterial cell lysis,or a lysogenic (non-lytic) cycle, which leaves the bacterial cellintact. The phage may be a naturally occurring phage isolate, or anengineered phage, including vectors, or nucleic acids that encode eithera partial phage genome (e.g., including at least all essential genesnecessary to carry out the life cycle of the phage inside a hostbacterium) or the full phage genome.

As used herein, the term “plant” refers to whole plants, plant organs,plant tissues, seeds, plant cells, seeds, and progeny of the same. Plantcells include, without limitation, cells from seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, roots,shoots, gametophytes, sporophytes, pollen, and microspores. Plant partsinclude differentiated and undifferentiated tissues including, but notlimited to the following: roots, stems, shoots, leaves, pollen, seeds,tumor tissue, and various forms of cells and culture (e.g., singlecells, protoplasts, embryos, and callus tissue). The plant tissue may bein a plant or in a plant organ, tissue, or cell culture. In addition, aplant may be genetically engineered to produce a heterologous protein orRNA, for example, of any of the modulating agents in the methods orcompositions described herein.

As used herein, the term “vector” refers to an insect that can carry ortransmit a human pathogen from a reservoir to a human. Exemplary vectorsinclude insects, such as those with piercing-sucking mouthparts, asfound in Hemiptera and some Hymenoptera and Diptera such as mosquitoes,bees, wasps, midges, lice, tsetse fly, fleas and ants, as well asmembers of the Arachnidae such as ticks and mites.

Other features and advantages of the invention will be apparent from thefollowing Detailed Description and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures are meant to be illustrative of one or more features,aspects, or embodiments of the invention and are not intended to belimiting.

FIG. 1A-1G show images of different antibiotic delivery systems. Firstinstar LSR-1 aphids were treated with different therapeutic solutions bydelivery through plants (FIG. 1A), leaf coating (FIG. 1B),microinjection (FIG. 1C), topical delivery (FIG. 1D), leaf perfusion andcutting (FIG. 1E), leaf perfusion and through plant (FIG. 1F), andcombination treatment of spraying both plant and aphid, and deliverythough plant (FIG. 1G).

FIG. 2A-2C show the delay in aphid development during rifampicintreatment in first instar LSR-1 aphids treated by delivery throughplants with three different conditions: artificial diet withoutessential amino acids (AD only), artificial diet without essential aminoacids with 100 μg/ml rifampicin (AD+Rif), and artificial diet with 100μg/ml rifampicin and essential amino acids (AD+Rif+EAA). FIG. 2A is aseries of graphs showing the percentage of living aphids at eachdevelopmental stage (sample size=33 aphids/group). FIG. 2B showsrepresentative images from each treatment taken at 12 days. Scale bars2.5 mm. FIG. 2C shows area measurements from aphid bodies showing thedrastic effect of rifampicin treatment. Adding back essential aminoacids partially rescues development defects.

FIG. 3 shows that rifampicin treatment resulted in aphid death. Survivalwas monitored daily for LSR-1 aphids treated by delivery through plantswith artificial diet without essential amino acids (AD only), artificialdiet without essential amino acids with 100 ug/ml rifampicin (AD+Rif),and artificial diet with 100 ug/ml rifampicin and (AD+Rif+EAA). Numberin parentheses represents number of aphids in each group. Statisticalsignificance was determined by Log-Rank Test and the followingstatistically significant differences were determined: AD only vs.AD+Rif, p<0.0001 and AD+Rif vs. AD+Rif+EAA, p=0.017.

FIG. 4 is a graph showing that rifampicin treatment resulted in loss ofreproduction in aphids. First instar LSR-1 aphids were treated bydelivery through plants with artificial diet without essential aminoacids (AD only), artificial diet without essential amino acids with 100ug/ml rifampicin (AD+Rif), and artificial diet with 100 ug/ml rifampicinand (AD+Rif+EAA) and the number of offspring produced each day afteraphid reached adulthood was measured. Shown is the mean number ofoffspring produced per day after aphid reached adulthood±S.D.

FIG. 5 is a graph showing that rifampicin treatment eliminatedendosymbiotic Buchnera. Symbiont titer was determined for the differentconditions at 7 days post-treatment. DNA from aphids was extracted andqPCR was performed to determine the ratio of Buchnera DNA to aphid DNA.Shown is the mean ratio of Buchnera DNA to aphid DNA±SD of 3 aphids pergroup. Statistically significant differences were determined using aone-way-ANOVA followed by Tukey's Post-Test; *, p<0.05.

FIGS. 6A and 6B show that rifampicin treatment delivered through leafcoating delayed aphid development. First instar eNASCO aphids weretreated by coating leaves with 100 μl of two different solutions:solvent control (0.025% Silwet L-77), and 50 μg/ml rifampicin. FIG. 6Ais a series of graphs showing the developmental stage over time for eachcondition. Shown is the percentage of living aphids at eachdevelopmental stage (sample size=20 aphids/group). FIG. 6B is a graphshowing area measurements from aphid bodies showing the drastic effectof rifampicin coated leaves on aphid size. Statistically significantdifferences were determined using a one-way-ANOVA followed by Tukey'sPost-Test; *, p<0.05.

FIG. 7 shows that rifampicin treatment delivered through leaf coatingresulted in aphid death. Survival was monitored daily for eNASCO aphidstreated by coating leaves with 100 μl of two different solutions:solvent control (Silwet L-77), and 50 μg/ml rifampicin. Treatmentaffects survival rate of aphids.

FIG. 8 shows that rifampicin treatment delivered through leaf coatingeliminated endosymbiotic Buchnera. Symbiont titer was determined for thetwo conditions at 6 days post-treatment. DNA from aphids was extractedand qPCR was performed to determine the ratio of Buchnera DNA to aphidDNA. Shown is the mean ratio of Buchnera DNA to aphid DNA±SD.Statistically significant differences were determined using aone-way-ANOVA followed by Tukey's Post-Test; *, p<0.05.

FIG. 9 is a graph showing rifampicin treatment by microinjectioneliminated endosymbiotic Buchnera. Symbiont titer was determined 4 dayspost-injection with the indicated conditions. Control sample is thesolvent, 0.025% Silwet L-77 described before. DNA from aphids wasextracted and qPCR was performed to determine the ratio of Buchnera DNAto aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA±SD.Statistically significant differences were determined using aone-way-ANOVA followed by Tukey's Post-Test; *, p<0.05.

FIG. 10 is a graph showing that rifampicin treatment delivered throughtopical treatment eliminated endosymbiotic Buchnera. Symbiont titer wasdetermined 3 days post-spraying with: solvent (silwet L-77) or therifampicin solution diluted in solvent. DNA from aphids was extractedand qPCR was performed to determine the ratio of Buchnera DNA to aphidDNA. Shown is the mean ratio of Buchnera DNA to aphid DNA±SD.Statistically significant differences were determined using aone-way-ANOVA followed by Tukey's Post-Test; *, p<0.05.

FIG. 11 shows a panel of graphs demonstrating that 1^(st) and 2^(nd)instar LSR-1 aphids were placed on leaves perfused with water plus foodcoloring or 50 μg/ml rifampicin in water plus food coloring.Developmental stage was measured over time for each condition. Shown isthe percentage of living aphids at each developmental stage (samplesize=74-81 aphids/group).

FIG. 12 shows a graph demonstrating survival of 1^(st) and 2^(nd) instarLSR-1 aphids placed on leaves perfused with water plus food coloring or50 μg/ml rifampicin in water plus food coloring. Number in parenthesesrepresents the number of aphids in each group. Statistical significancewas determined by Log-Rank Test.

FIG. 13 shows a graph demonstrating symbiont titer determined 8 dayspost-treatment with leaves perfused with water and food coloring orrifampicin plus water and food coloring. DNA from aphids was extractedand qPCR was performed to determine the ratio of Buchnera DNA to aphidDNA. Shown is the mean ratio of Buchnera DNA to aphid DNA±SD. Number inbox indicates the median of the experimental group.

FIG. 14 shows a panel of graphs demonstrating 1^(st) and 2^(nd) instarLSR-1 aphids treated via leaf injection and through the plant with waterplus food coloring or 100 μg/ml rifampicin in water plus food coloring.Developmental stage was measured over time for each condition. Shown isthe percentage of living aphids at each developmental stage (samplesize=49-50 aphids/group).

FIG. 15 is a graph demonstrating survival of 1^(st) and 2^(nd) instarLSR-1 aphids placed on leaves perfused and treated with water plus foodcoloring or 100 μg/ml rifampicin in water plus food coloring. Number inparentheses represents the number of aphids in each group. A Log-RankTest was performed and determined that there were no statisticallysignificant differences between groups.

FIGS. 16A and 16B are graphs showing symbiont titer determined 6 (16A)and 8 (16B) days post-treatment in aphids feeding on leaves perfused andtreated with water and food coloring or rifampicin plus water and foodcoloring. DNA was extracted from aphids and qPCR was performed todetermine the ratio of Buchnera DNA to aphid DNA. Shown is the meanratio of Buchnera DNA to aphid DNA±SD. Number in box indicates themedian of the experimental group.

FIG. 17 is a panel of graphs showing that 1^(st) and 2^(nd) instar LSR-1aphids were treated with control solutions (water and Silwet L-77) or acombination of treatments with 100 μg/ml rifampicin. Developmental stagewas measured over time for each condition. Shown is the percentage ofliving aphids at each developmental stage (sample size=76-80aphids/group).

FIG. 18 is a graph showing 1st and 2nd instar LSR-1 aphids were treatedwith control solutions of a combination of treatments containingrifampicin. Number in parentheses represents the number of aphids ineach group. A Log-Rank Test was performed and determined that there wereno statistically significant differences between groups.

FIG. 19 is a graph showing symbiont titer determined at 7 dayspost-treatment with control or rifampicin solutions. DNA from aphids wasextracted and qPCR was performed to determine the ratio of Buchnera DNAto aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA±SD.Number in box indicates the median of the experimental group.Statistically significant differences were determined by t-test.

FIG. 20 is an image showing the chitosan delivery system. A. pisumaphids were treated with a therapeutic solution by delivery through leafperfusion and through the plants as shown.

FIG. 21 is a panel of graphs showing that chitosan treatment resulted indelayed aphid development. First and second instar A. pisum aphids weretreated by delivery through plants and leaf perfusion with the controlsolution (Water), and 300 ug/ml chitosan in water. Developmental stagewas monitored throughout the experiment. Shown are the percent of aphidsat each developmental stage (1st instar, 2nd instar, 3rd instar, 4thinstar, 5th instar, or 5R which represents a reproducing 5th instar) pertreatment group.

FIG. 22 is a graph showing there was a decrease in insect survival upontreatment with chitosan. First and second instar A. pisum aphids weretreated by delivery through plants and leaf perfusion with just water orchitosan solution and survival was monitored daily over the course ofthe experiment. Number in parentheses represents the total number ofaphids in the treatment group.

FIG. 23 is a graph showing treatment with chitosan reduced endosymbioticBuchnera. First and second instar A. pisum aphids were treated bydelivery through plants and leaf perfusion with water or 300 ug/mlchitosan in water. At 8 days post-treatment, DNA from aphids wasextracted and qPCR was performed to determine the ratio of Buchnera DNAto aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA±SD of6 aphids/group. The median value for each group is shown in box.

FIG. 24 is a panel of graphs showing treatment with nisin resulted indelayed aphid development. First and second instar LSR-2 A. pisum aphidswere treated with water (control) or 1.6 or 7 mg/ml nisin via deliveryby leaf injection and through the plant and development was measuredover time. Shown are the percent of aphids at each life stage (1st, 2nd,3rd, 4th, 5th, and 5R (reproducing 5th) instar) at the indicated timepoint. N=56-59 aphids/group.

FIG. 25 is a graph showing there was a dose dependent decrease in insectsurvival upon treatment with nisin. First and second instar LSR-1 A.pisum aphids were treated with water (control) or 1.6 or 7 mg/ml nisinvia delivery by leaf injection and through the plant and survival wasmonitored over time. Number in parentheses indicates the number ofaphids/group. Statistically significant differences were determined byLog Rank (Mantel-Cox) test.

FIG. 26 is a graph showing treatment with nisin reduced endosymbioticBuchnera. First and second instar LSR-1 A. pisum aphids were treatedwith water (control) or 1.6 mg/ml nisin via delivery by leaf injectionand through the plant and DNA was extracted from select aphids at eightdays post-treatment and used for qPCR to determine Buchnera copynumbers. Shown are the mean Buchnera/aphid ratios for eachtreatment+/−SEM. Number in the box above each experimental groupindicates the median value for that group. Each data point represents asingle aphid.

FIG. 27 is a panel of graphs showing treatment with levulinic acidresulted in delayed aphid development. First and second instar eNASCO A.pisum aphids were treated with water (control) or 0.03 or 0.3% levulinicacid via delivery by leaf injection and through the plant anddevelopment was measured over time. Shown are the percent of aphids ateach life stage (1^(st), 2^(nd), 3^(rd), 4^(th), and 5^(th) instar) atthe indicated time point. N=57-59 aphids/group.

FIG. 28 is a graph showing there was a decrease in insect survival upontreatment with levulinic acid. First and second instar eNASCO A. pisumaphids were treated with water (control) or 0.03 or 0.3% levulinic acidvia delivery by leaf injection and through the plant and survival wasmonitored over time. N=57-59 aphids/group. Statistically significantdifferences were determined by Log Rank (Mantel-Cox) test; **, p<0.01.

FIG. 29 is a panel of graphs showing treatment with levulinic acidreduced endosymbiotic Buchnera. First and second instar eNASCO A. pisumaphids were treated with water (control) or 0.03 or 0.3% levulinic acidvia delivery by leaf injection and through the plant and DNA wasextracted from select aphids at seven and eleven days post-treatment andused for qPCR to determine Buchnera copy numbers. Shown are the meanBuchnera/aphid ratios for each treatment+/−SEM. Statisticallysignificant differences were determined by One-way ANOVA and Dunnett'sMultiple Comparison Test; *, p<0.05. Each data point represents a singleaphid.

FIGS. 30A and 30B show graphs demonstrating that gossypol treatmentresulted in delayed aphid development. First and second instar A. pisumaphids were treated by delivery through plants with artificial dietwithout essential amino acids (AD only), and artificial diet withoutessential amino acids with different concentrations of gossypol (0.05%,0.25% and 0.5%). Developmental stage was monitored throughout theexperiment. FIG. 30A is a series of graphs showing the mean number ofaphids at each developmental stage (1st instar, 2nd instar, 3rd instar,4th instar, 5th instar, or 5R which represents a reproducing 5th instar)per treatment group. At the indicated time, aphids were imaged and theirsize was determined using Image J. FIG. 30B is a graph showing the meanaphid area±SD of artificial diet treated (Control) or gossypol treatedaphids. Statistical significance was determined using a One-Way ANOVAfollowed by Tukey's post-test. *, p<0.05. **, p<0.01.

FIG. 31 is a graph showing a dose-dependent decrease in survival ofaphids upon treatment with the allelochemical gossypol. First and secondinstar A. pisum aphids were treated by delivery through plants withartificial diet without essential amino acids (AD no EAA), artificialdiet without essential amino acids with 0.5% gossypol acetic acid (0.5%gossypol), artificial diet without essential amino acids with 0.25%gossypol acetic acid (0.25% gossypol), and artificial diet withoutessential amino acids and 0.05% gossypol acetic acid (0.05% gossypol)and survival was monitored daily over the course of the experiment.Number in parentheses represents the essential amino acids number ofaphids in each group. Statistically significant differences weredetermined by Log-Rank test and AD no EAA and 0.5% gossypol aresignificantly different, p=0.0002.

FIGS. 32A and 32B are two graphs showing that treatment with 0.25%gossypol resulted in decreased fecundity. First and second instar A.pisum aphids were treated by delivery through plants with artificialdiet without essential amino acids (AD5-2 no EAA), or artificial dietwithout essential amino acids with 0.25% gossypol acetic acid (AD5-2 noEAA+0.25% gossypol), and fecundity was determined throughout the timecourse of the experiment. FIG. 32A shows the mean day±SD at which aphidsbegan producing offspring was measured and gossypol treatment delayedproduction of offspring. FIG. 32B shows the mean number of offspringproduced after the aphid began a reproducing adult±SD was measured andgossypol treatment results in decreased number of offspring produced.Each data point represents one aphid.

FIG. 33 is a graph showing that treatment with different concentrationsof gossypol reduced endosymbiotic Buchnera. First and second instar A.pisum aphids were treated by delivery through plants with artificialdiet without essential amino acids (Control)) or artificial diet withoutessential amino acids with 0.5%, 0.25%, or 0.05% gossypol. At 5 or 13days post-treatment, DNA from aphids was extracted and qPCR wasperformed to determine the ratio of Buchnera DNA to aphid DNA. Shown isthe mean ratio of Buchnera DNA to aphid DNA±SD of 2-6 aphids/group.Statistically significant differences were determined by UnpairedT-test; *, p<0.05.

FIG. 34 is a graph showing that microinjection of gossypol resulted indecreased Buchnera levels in aphids. A. pisum LSR-1 aphids<3rd instarstage (nymphs) were injected with 20 nl of artificial diet withoutessential amino acids (AD) or artificial diet without essential aminoacids with 0.05% gossypol (gossypol (0.05%)). Three days afterinjection, DNA was extracted from aphids and Buchnera levels wereassessed by qPCR. Shown are the mean ratios of Buchnera/aphid DNA±SD.Each data point represents one aphid.

FIG. 35 is a panel of graphs showing Trans-cinnemaldehyde treatmentresulted in delayed aphid development. First and second instar A. pisumaphids were treated by delivery through plants with water and water withdifferent concentrations of trans-cinnemaldehyde (TC, 0.05%, 0.5%, and5%). Developmental stage was monitored throughout the experiment. Shownare the mean number of aphids at each developmental stage (1st instar,2nd instar, 3rd instar, 4th instar, 5th instar, or 5R which represents areproducing 5th instar) per treatment group. N=40-49 aphids/experimentalgroup.

FIG. 36 is a graph showing there was a dose-dependent decrease insurvival upon treatment the natural antimicrobial trans-cinnemaldehyde.First and second instar A. pisum aphids were treated by delivery throughplants with water and water with different concentrations oftrans-cinnemaldehyde (TC, 0.05%, 0.5%, and 5%). Survival was monitoredthroughout the course of the treatment. Statistically significantdifferences were determined by Log-Rank test. N=40-49 aphids/group.

FIG. 37 is a graph showing treatment with different concentrations oftrans-cinnemaldehyde reduced endosymbiotic Buchnera. First and secondinstar A. pisum aphids were treated by delivery through plants withwater and water with different concentrations of trans-cinnemaldehyde(0.05%, 0.5%, and 5%). At 3 days post-treatment, DNA from aphids wasextracted and qPCR was performed to determine the ratio of Buchnera DNAto aphid DNA. Shown is the mean ratio of Buchnera DNA to aphid DNA±SD of2-11 aphids/group. The median of each treatment group is shown in thebox above the data points. Statistically significant differences weredetermined by Unpaired T-test; *, p<0.05. There was a statisticallysignificant difference between the water control and the 0.5%trans-cinnemaldehyde group.

FIG. 38 is a panel of graphs showing treatment with scorpion peptideUy192 resulted in delayed aphid development. First and second instar A.pisum aphids were treated by delivery through plants and leaf perfusionwith the control solution (water), and 100 ug/ml Uy192 in water. a)developmental stage was monitored throughout the experiment. Shown arethe percent of aphids at each developmental stage (1st instar, 2ndinstar, 3rd instar, 4th instar, 5th instar, or 5R which represents areproducing 5th instar) per treatment group.

FIG. 39 is a graph showing there was a decrease in insect survival upontreatment with the scorpion AMP Uy192. First and second instar A. pisumaphids were treated by delivery through plants and leaf perfusion withjust water or Uy192 solution and survival was monitored daily over thecourse of the experiment. Number in parentheses represents the totalnumber of aphids in the treatment group.

FIG. 40 is a graph showing treatment with Uy192 reduced endosymbioticBuchnera. First and second instar A. pisum aphids were treated bydelivery through plants and leaf perfusion with water or 100 ug/ml Uy192in water, at 8 days post-treatment, DNA from aphids was extracted andqPCR was performed to determine the ratio of Buchnera DNA to aphid DNA.Shown is the mean ratio of Buchnera DNA to aphid DNA±SD of 2-6aphids/group. The median value for each group is shown in box.

FIG. 41 is a graph showing a decrease in survival in aphidsmicroinjected with scorpion peptides D10 and D3. LSR-1 A. pisum aphidswere microinjected with water (control) or with 100 ng of eitherscorpion peptide D3 or D10. After injection, aphids were released tofava bean leaves and survival was monitored throughout the course of theexperiment. The number in parentheses indicates the number of aphids ineach experimental treatment group.

FIG. 42 is a graph showing a decrease in endosymbiont titers uponinjection with scorpion peptides D3 and D10. LSR-1 A. pisum aphids weremicroinjected with water (control) or with 100 ng of either scorpionpeptide D3 or D10. After injection, aphids were released to fava beanleaves and at 5 days post-treatment, DNA was extracted from theremaining living aphids and qPCR was performed to determine the ratio ofBuchnera/aphid DNA. Shown are the mean±SD of each treatment group. N=2-9aphids/group. The number above each treatment group in the boxrepresents the median of the dataset.

FIG. 43 is a graph showing a decrease in insect survival upon treatmentwith a cocktail of scorpion AMPs. First and second instar eNASCO aphidswere treated by delivery through leaf perfusion and through plants witha cocktail of scorpion peptides (40 μg/ml of each of Uy17, D3, UyCt3,and D10) and survival was monitored over the course of the experiment.The number in parentheses represents the number of aphids in eachtreatment group.

FIG. 44 is a panel of graphs showing treatment with scorpion peptidefused to a cell penetrating peptide resulted in delayed aphiddevelopment. First instar LSR-2 A. pisum aphids were treated with water(control) or 100 μg/ml Uy192+CPP+FAM via delivery by leaf injection andthrough the plant and development was measured over time. Shown are thepercent of aphids at each life stage (1st, 2nd, 3rd, 4th, 5th, and 5R(reproducing 5th) instar) at the indicated time point. N=90aphids/group.

FIG. 45 is a graph showing treatment of aphids with a scorpion peptidefused to a cell penetrating peptide increased mortality. First instarLSR-1 A. pisum aphids were treated with water or 100 μg/ml UY192+CPP+FAM(peptide) in water delivered by leaf injection and through the plant.Survival was monitored over time. The number in parentheses indicatesthe number of aphids/group. Statistically significant differences weredetermined by Log Rank (Mantel-Cox) test and there is a significantdifference between the two experimental groups (p=0.0036).

FIG. 46 is a graph showing treatment with Uy192+CPP+FAM reducedendosymbiotic Buchnera. First instar LSR-1 A. pisum aphids were treatedwith water or 100 μg/ml Uy192+CPP+FAM (peptide) in water delivered byleaf injection and through the plant. DNA was extracted from selectaphids at five days post-treatment and used for qPCR to determineBuchnera copy numbers. Shown are the mean Buchnera/aphid ratios for eachtreatment+/−SEM. Number in the box above each experimental groupindicates the median value for that group. Each data point represents asingle aphid. Statistically significant differences were determined byStudent's T-test; ****, p<0.0001.

FIG. 47 is a panel of images showing Uy192+CPP+FAM penetratedbacteriocyte membranes. Bacteriocytes were dissected from the aphids andincubated with 250 μg/ml of the Uy192+CPP+FAM peptide for 30 min. Uponwashing and imaging, the Uy192+CPP+FAM can be seen at high quantitiesinside the bacteriocytes.

FIG. 48A and FIG. 48B are a panel of graphs showing pantothenoltreatment delayed aphid development. First instar and second eNASCOaphids were treated by delivery through plants with three differentconditions: artificial diet without essential amino acids (AD no EAA),artificial diet without essential amino acids with 10 uM pantothenol (10uM pantothenol), and artificial diet without essential amino acids with100 uM pantothenol (100 uM pantothenol), artificial diet withoutessential amino acids with 100 uM pantothenol, and artificial dietwithout essential amino acids with 10 uM pantothenol. FIG. 48A showsdevelopmental stage monitored over time for each condition. FIG. 48Bshows relative area measurements from aphid bodies showing the drasticeffect of pantothenol treatment.

FIG. 49 is a graph showing that treatment with pantothenol increasedaphid mortality. Survival was monitored daily for eNASCO aphids treatedby delivery through plants with artificial diet without essential aminoacids, or artificial diet without essential amino acids containing 10 or100 uM pantothenol. Number in parentheses represents number of aphids ineach group.

FIGS. 50A, 50B, and 50C are a panel of graphs showing Pantothenoltreatment resulted in loss of reproduction. First and second instareNASCO aphids were treated by delivery through plants with artificialdiet without essential amino acids or with artificial diet withoutessential amino acids with 10 or 100 uM pantothenol. FIG. 50A shows thefraction of aphids surviving to maturity and reproducing. FIG. 50B showsthe mean day aphids in each group began reproducing. Shown is the meanday an aphid began reproducing ±SD. FIG. 50C shows the mean number ofoffspring produced per day after an aphid began reproducing. Shown arethe mean number of offspring/day±SD.

FIG. 51 is a graph showing Pantothenol treatment did not affectendosymbiotic Buchnera. Symbiont titer was determined for the differentconditions at 8 days post-treatment. DNA from aphids was extracted andqPCR was performed to determine the ratio of Buchnera DNA to aphid DNA.Shown is the mean ratio of Buchnera DNA to aphid DNA±SD of 6 aphids pergroup.

FIG. 52 is a panel of graphs showing Pantothenol treatment deliveredthrough plants did not affect aphid development. First instar eNASCOaphids were treated by coating leaves with 100 μl of two differentsolutions: solvent control (0.025% Silwet L-77), and 10 uM pantothenoland the developmental stage was measured over time for each condition.Shown is the percentage of living aphids at each developmental stage(sample size=20 aphids/group).

FIG. 53 is a graph showing Pantothenol treatment delivered through leafcoating resulted in aphid death. Survival was monitored daily for eNASCOaphids treated by coating leaves with 100 μl of two different solutions:solvent control (Silwet L-77), and 10 uM pantothenol. Treatment affectssurvival rate of aphids. Sample size=20 aphids/group. Log-Rank MantelCox test was used to determine whether there were statisticallysignificant differences between groups and identified that the two groupare significantly different (p=0.0019).

FIGS. 54A and 54B are a panel of graphs showing treatment with acocktail of amino acid analogs delayed aphid development. First instarLSR-1 aphids were treated by delivery through leaf perfusion and throughplants with water or a cocktail of amino acid analogs in water (AAcocktail). FIG. 54A shows the developmental stage measured over time foreach condition. Shown are the percentage of living aphids at eachdevelopmental stage. FIG. 54B shows the area measurements from aphidbodies showing the drastic effect of treatment with an amino acid analogcocktail (AA cocktail). Statistically significant differences weredetermined using a Student's T-test; ****, p<0.0001.

FIG. 55 is a graph showing treatment with a cocktail of amino acidanalogs eliminated endosymbiotic Buchnera. Symbiont titer was determinedfor the different conditions at 6 days post-treatment. DNA from aphidswas extracted and qPCR was performed to determine the ratio of BuchneraDNA to aphid DNA. Shown are the mean ratios of Buchnera DNA to aphidDNA±SD of 19-20 aphids per group. Each data point represents anindividual aphid. Statistically significant differences were determinedusing a Student's T-test; *, p<0.05.

FIGS. 56A and 56B is a panel of graphs showing treatment with acombination of three agents delayed aphid development. First instarLSR-1 aphids were treated by delivery through leaf perfusion and throughplants with water or a combination of three agents in water(Pep-Rif-Chitosan). FIG. 56A shows the developmental stage measured overtime for each condition. Shown are the percentage of living aphids ateach developmental stage. FIG. 56B shows the area measurements fromaphid bodies showing the drastic effect of treatment with a combinationof three treatments (Pep-Rif-Chitosan). Statistically significantdifferences were determined using a Student's T-test; ****, p<0.0001.

FIG. 57 is a graph showing treatment with a combination of a peptide,antibiotic, and natural antimicrobial agent increased aphid mortality.LSR-1 aphids were treated with water or a combination of threetreatments (Pep-Rif-Chitosan) and survival was monitored daily aftertreatment.

FIG. 58 is a graph showing treatment with a combination of a peptide,antibiotic, and natural antimicrobial agent eliminated endosymbioticBuchnera. Symbiont titer was determined for the different conditions at6 days post-treatment. DNA from aphids was extracted and qPCR wasperformed to determine the ratio of Buchnera DNA to aphid DNA. Shown arethe mean ratios of Buchnera DNA to aphid DNA±SD of 20-21 aphids pergroup. Each data point represents an individual aphid.

FIGS. 59A and 59B are a panel of images showing ciprofloxacin coated andpenetrated corn kernels. Corn kernels were soaked in water (noantibiotic) or the indicated concentration of ciprofloxacin in water andwhole kernels or kernel were tested to see whether they can inhibit thegrowth of E. coli DH5a. FIG. 59A shows bacterial growth in the presenceof a corn kernel soaked in water without antibiotics and FIG. 59B showsthe inhibition of bacterial growth when whole or half corn kernels thathave been soaked in antibiotics are placed on a plate spread with E.coli.

FIG. 60 is a graph showing that adult S. zeamais weevils were treatedwith ciprofloxacin (250 ug/ml or 2.5 mg/ml) or mock treated with water.After 18 days of treatment, genomic DNA was isolated from weevils andthe amount of Sitophilus primary endosymbiont was determined by qPCR.Shown is the mean±SEM of each group. Each data point represents oneweevil. The median of each group is listed above the dataset.

FIGS. 61A and 61B are graphs showing weevil development after treatmentwith ciprofloxacin. FIG. 61A shows individual corn kernels cut open 25days after adults were removed from one replicate each of the initialcorn kernels soaked/coated with water (control) or ciprofloxacin (250ug/ml or 2.5 mg/ml) and examined for the presence of larvae, pupae, oralmost fully developed (adult) weevils. Shown is the percent of eachlife stage found in kernels from each treatment group. The total numberof offspring found in the kernels from each treatment group is indicatedabove each dataset. FIG. 61B shows genomic DNA isolated from offspringdissected from corn kernels from the control (water) and 2.5 mg/mlciprofloxacin treatment groups and qPCR was done to measure the amountof Sitophilus primary endosymbiont present. Shown are the mean±SD foreach group. Statistically significant differences were determined byunpaired t-test; ***, p≤0.001.

FIGS. 62A and 62B are graphs showing the two remaining replicates ofcorn kernels mock treated (water) or treated with 250 ug/ml or 2.5 mg/mlciprofloxacin monitored for the emergence of offspring after matingpairs were removed (at 7 days post-treatment). FIG. 62A shows the meannumber of newly emerged weevils over time±SD for each treatment group.FIG. 62B shows the mean number±SEM of emerged weevils for each treatmentgroup at 43 days after mating pairs were removed.

FIG. 63 is a graph showing rifampicin and doxycycline treatment resultedin mite mortality. Survival was monitored daily for untreatedtwo-spotted spider mites and mites treated with 250 μg/ml rifampicin and500 μg/ml doxycycline in 0.025% Silwet L-77.

FIG. 64 is a panel of graphs showing the results of a Seahorse fluxassay for bacterial respiration. Bacteria were grown to logarithmicphase and loaded into Seahorse XFe96 plates for temporal measurements ofoxygen consumption rate (OCR) and extracellular acidification rate(ECAR) as described in methods. Treatments were injected into the wellsafter approximately 20 minutes and bacteria were monitored to detectchanges in growth. Rifampicin=100 μg/mL; Chloramphenicol=25 μg/mL;Phages (T7 for E. coli and ϕSmVL-C1 for S. marcescens) were lysatesdiluted either 1:2 or 1:100 in SM Buffer. The markers on each line aresolely provided as indicators of the condition to which each linecorresponds, and are not indicative of data points.

FIG. 65 is a graph showing phage against S. marcescens reduced flymortality. Flies that were pricked with S. marcescens were all deadwithin a day, whereas a sizeable portion of the flies that were prickedwith both S. marcescens and the phage survived for five days after thetreatment. Almost all of the control flies which were not treated inanyway survived till the end of the experiment. Log-rank test was usedto compare the curves for statistical significance, asterisk denotesp<0.0001.

DETAILED DESCRIPTION

Provided herein are methods and compositions useful for human health,e.g., for altering a level, activity, or metabolism of one or moremicroorganisms resident in a host insect (e.g., arthropod, e.g., insect,e.g., a human pathogen vector, e.g., mosquito, mite, louse, or tick),the alteration resulting in a decrease in the fitness of the host. Theinvention features a composition that includes a modulating agent (e.g.,phage, peptide, small molecule, antibiotic, or combinations thereof)that can alter the host's microbiota in a manner that is detrimental tothe host. By disrupting microbial levels, microbial activity, microbialmetabolism, or microbial diversity, the modulating agent describedherein may be used to decrease the fitness of a variety of insects thatcarry vector-borne pathogens that cause disease in humans.

The methods and compositions described herein are based in part on theexamples provided herein, which illustrate how modulating agents, forexample antibiotics (e.g., oxytetracycline, doxycycline, or acombination thereof) can be used to target symbiotic microorganisms in ahost (e.g., endosymbionts e.g., endosymbiotic Wolbachia in mosquitos orRickettsia in ticks) in insect vectors of human pathogens, to decreasethe fitness of the host by altering the level, activity, or metabolismof the microorganisms within the hosts. Oxytetracycline and doxycyclineare representative examples of antibiotics useful for this purpose. Onthis basis the present disclosure describes a variety of differentapproaches for the use of agents that alter a level, activity, ormetabolism of one or more microorganisms resident in a host (e.g., avector of a human pathogen, e.g., a mosquito, mite, louse or a tick) thealteration resulting in a decrease in the host's fitness.

I. Hosts

i. Insects

The methods and compositions provided herein may be used with any insecthost that is considered a vector for a pathogen that is capable ofcausing disease in humans

For example, the insect host may include, but is not limited to thosewith piercing-sucking mouthparts, as found in Hemiptera and someHymenoptera and Diptera such as mosquitoes, bees, wasps, midges, lice,tsetse fly, fleas and ants, as well as members of the Arachnidae such asticks and mites; order, class or family of Acarina (ticks and mites)e.g. representatives of the families Argasidae, Dermanyssidae, Ixodidae,Psoroptidae or Sarcoptidae and representatives of the species Amblyommaspp., Anocenton spp., Argas spp., Boophilus spp., Cheyletiella spp.,Chorioptes spp., Demodex spp., Dermacentor spp., Denmanyssus spp.,Haemophysalis spp., Hyalomma spp., Ixodes spp., Lynxacarus spp.,Mesostigmata spp., Notoednes spp., Ornithodoros spp., Ornithonyssusspp., Otobius spp., otodectes spp., Pneumonyssus spp., Psoroptes spp.,Rhipicephalus spp., Sancoptes spp., or Trombicula spp.; Anoplura(sucking and biting lice) e.g. representatives of the species Bovicolaspp., Haematopinus spp., Linognathus spp., Menopon spp., Pediculus spp.,Pemphigus spp., Phylloxera spp., or Solenopotes spp.; Diptera (flies)e.g. representatives of the species Aedes spp., Anopheles spp.,Calliphora spp., Chrysomyia spp., Chrysops spp., Cochliomyia spp., Cw/exspp., Culicoides spp., Cuterebra spp., Dermatobia spp., Gastrophilusspp., Glossina spp., Haematobia spp., Haematopota spp., Hippobosca spp.,Hypoderma spp., Lucilia spp., Lyperosia spp., Melophagus spp., Oestrusspp., Phaenicia spp., Phlebotomus spp., Phormia spp., Acari (sarcopticmange) e.g., Sarcoptidae spp., Sarcophaga spp., Simulium spp., Stomoxysspp., Tabanus spp., Tannia spp. or Zzpu/alpha spp.; Mallophaga (bitinglice) e.g. representatives of the species Damalina spp., Felicola spp.,Heterodoxus spp. or Trichodectes spp.; or Siphonaptera (winglessinsects) e.g. representatives of the species Ceratophyllus spp.,Xenopsylla spp; Cimicidae (true bugs) e.g. representatives of thespecies Cimex spp., Tritominae spp., Rhodinius spp., or Triatoma spp.

In some instances, the insect is a blood-sucking insect from the orderDiptera (e.g., suborder Nematocera, e.g., family Colicidae). In someinstances, the insect is from the subfamilies Culicinae, Corethrinae,Ceratopogonidae, or Simuliidae. In some instances, the insect is of aCulex spp., Theobaldia spp., Aedes spp., Anopheles spp., Aedes spp.,Forciponiyia spp., Culicoides spp., or Helea spp.

In certain instances, the insect is a mosquito. In certain instances,the insect is a tick. In certain instances, the insect is a mite. Incertain instances, the insect is a biting louse.

ii. Host Fitness

The methods and compositions provided herein may be used to decrease thefitness of any of the hosts described herein. The decrease in fitnessmay arise from any alterations in microorganisms resident in the host,wherein the alterations are a consequence of administration of amodulating agent and have detrimental effects on the host.

In some instances, the decrease in host fitness may manifest as adeterioration or decline in the physiology of the host (e.g., reducedhealth or survival) as a consequence of administration of a modulatingagent. In some instances, the fitness of an organism may be measured byone or more parameters, including, but not limited to, reproductiverate, lifespan, mobility, fecundity, body weight, metabolic rate oractivity, or survival in comparison to a host organism to which themodulating agent has not been administered. For example, the methods orcompositions provided herein may be effective to decrease the overallhealth of the host or to decrease the overall survival of the host. Insome instances, the decreased survival of the host is about 2%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%greater relative to a reference level (e.g., a level found in a hostthat does not receive a modulating agent). In some instances, themethods and compositions are effective to decrease host reproduction(e.g., reproductive rate) in comparison to a host organism to which themodulating agent has not been administered. In some instances, themethods and compositions are effective to decrease other physiologicalparameters, such as mobility, body weight, life span, fecundity, ormetabolic rate, by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, or greater than 100% relative to a reference level (e.g., alevel found in a host that does not receive a modulating agent).

In some instances, the decrease in host fitness may manifest as adecrease in the production of one or more nutrients in the host (e.g.,vitamins, carbohydrates, amino acids, or polypeptides). In someinstances, the methods or compositions provided herein may be effectiveto decrease the production of nutrients in the host (e.g., vitamins,carbohydrates, amino acids, or polypeptides) by about 2%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% relativeto a reference level (e.g., a level found in a host that does notreceive a modulating agent). In some instances, the methods orcompositions provided herein may decrease nutrients in the host bydecreasing the production of nutrients by one or more microorganisms(e.g., endosymbiont) in the host in comparison to a host organism towhich the modulating agent has not been administered.

In some instances, the decrease in host fitness may manifest as anincrease in the host's sensitivity to a pesticidal agent (e.g., apesticide listed in Table 12) and/or a decrease in the host's resistanceto a pesticidal agent (e.g., a pesticide listed in Table 12) incomparison to a host organism to which the modulating agent has not beenadministered. In some instances, the methods or compositions providedherein may be effective to increase the host's sensitivity to apesticidal agent (e.g., a pesticide listed in Table 12) by about 2%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%relative to a reference level (e.g., a level found in a host that doesnot receive a modulating agent). The pesticidal agent may be anypesticidal agent known in the art, including insecticidal agents. Insome instances, the methods or compositions provided herein may increasethe host's sensitivity to a pesticidal agent (e.g., a pesticide listedin Table 12) by decreasing the host's ability to metabolize or degradethe pesticidal agent into usable substrates.

In some instances, the decrease in host fitness may manifest as anincrease in the host's sensitivity to an allelochemical agent and/or adecrease in the host's resistance to an allelochemical agent incomparison to a host organism to which the modulating agent has not beenadministered. In some instances, the methods or compositions providedherein may be effective to decrease the host's resistance to anallelochemical agent by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, or greater than 100% relative to a reference level(e.g., a level found in a host that does not receive a modulatingagent). In some instances, the allelochemical agent is caffeine,soyacystatin N, monoterpenes, diterpene acids, or phenolic compounds. Insome instances, the methods or compositions provided herein may increasethe host's sensitivity to an allelochemical agent by decreasing thehost's ability to metabolize or degrade the allelochemical agent intousable substrates in comparison to a host organism to which themodulating agent has not been administered.

In some instances, the methods or compositions provided herein may beeffective to decease the host's resistance to parasites or pathogens(e.g., fungal, bacterial, or viral pathogens or parasites) in comparisonto a host organism to which the modulating agent has not beenadministered. In some instances, the methods or compositions providedherein may be effective to decrease the host's resistance to a pathogenor parasite (e.g., fungal, bacterial, or viral pathogens; or parasiticmites) by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, or greater than 100% relative to a reference level (e.g., a levelfound in a host that does not receive a modulating agent).

In some instances, the decrease in host fitness may manifest as otherfitness disadvantages, such as decreased tolerance to certainenvironmental factors (e.g., a high or low temperature tolerance),decreased ability to survive in certain habitats, or a decreased abilityto sustain a certain diet in comparison to a host organism to which themodulating agent has not been administered. In some instances, themethods or compositions provided herein may be effective to decreasehost fitness in any plurality of ways described herein. Further, themodulating agent may decrease host fitness in any number of hostclasses, orders, families, genera, or species (e.g., 1 host species, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 200, 250, 500, or more host species). In some instances, themodulating agent acts on a single host class, order, family, genus, orspecies.

Host fitness may be evaluated using any standard methods in the art. Insome instances, host fitness may be evaluated by assessing an individualhost. Alternatively, host fitness may be evaluated by assessing a hostpopulation. For example, a decrease in host fitness may manifest as adecrease in successful competition against other insects, therebyleading to a decrease in the size of the host population.

iii. Host Insects in Disease Transmission

By decreasing the fitness of host insects that carry human pathogens,the modulating agents provided herein are effective to reduce the spreadof vector-borne diseases. The modulating agent may be delivered to theinsects using any of the formulations and delivery methods describedherein, in an amount and for a duration effective to reduce transmissionof the disease, e.g., reduce vertical or horizontal transmission betweenvectors and/or reduce transmission to humans. For example, themodulating agent described herein may reduce vertical or horizontaltransmission of a vector-borne pathogen by about 2%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a hostorganism to which the modulating agent has not been administered. As ananother example, the modulating agent described herein may reducevectorial competence of an insect vector by about 2%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, or more in comparison to a hostorganism to which the modulating agent has not been administered.

Non-limiting examples of diseases that may be controlled by thecompositions and methods provided herein include diseases caused byTogaviridae viruses (e.g., Chikungunya, Ross River fever, Mayaro,Onyon-nyong fever, Sindbis fever, Eastern equine enchephalomyeltis,Wesetern equine encephalomyelitis, Venezualan equine encephalomyelitis,or Barmah forest); diseases caused by Flavivirdae viruses (e.g., Denguefever, Yellow fever, Kyasanur Forest disease, Omsk haemorrhagic fever,Japaenese encephalitis, Murray Valley encephalitis, Rocio, St. Louisencephalitis, West Nile encephalitis, or Tick-borne encephalitis);diseases caused by Bunyaviridae viruses (e.g., Sandly fever, Rift Valleyfever, La Crosse encephalitis, California encephalitis, Crimean-Congohaemorrhagic fever, or Oropouche fever); disease caused by Rhabdoviridaeviruses (e.g., Vesicular stomatitis); disease caused by Orbiviridae(e.g., Bluetongue); diseases caused by bacteria (e.g., Plague,Tularaemia, Q fever, Rocky Mountain spotted fever, Murine typhus,Boutonneuse fever, Queensland tick typhus, Siberian tick typhus, Scrubtyphus, Relapsing fever, or Lyme disease); or diseases caused byprotozoa (e.g., Malaria, African trypanosomiasis, Nagana, Chagasdisease, Leishmaniasis, Piroplasmosis, Bancroftian filariasis, orBrugian filariasis).

II. Target Microorganisms

The microorganisms targeted by the modulating agent described herein mayinclude any microorganism resident in or on the host, including, but notlimited to, any bacteria and/or fungi described herein. Microorganismsresident in the host may include, for example, symbiotic (e.g.,endosymbiotic microorganisms that provide beneficial nutrients orenzymes to the host), commensal, pathogenic, or parasiticmicroorganisms. An endosymbiotic microorganism may be a primaryendosymbiont or a secondary endosymbiont. A symbiotic microorganism(e.g., bacteria or fungi) may be an obligate symbiont of the host or afacultative symbiont of the host. Microorganisms resident in the hostmay be acquired by any mode of transmission, including vertical,horizontal, or multiple origins of transmission.

i. Bacteria

Exemplary bacteria that may be targeted in accordance with the methodsand compositions provided herein, include, but are not limited to,Xenorhabdus spp, Photorhabdus spp, Candidatus spp, Buchnera spp,Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp,Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidusspp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp,Xylella spp, Erwinia spp, Agrobacterium spp, Bacillus spp, Paenibacillusspp, Streptomyces spp, Micrococcus spp, Corynebacterium spp, Acetobacterspp, Cyanobacteria spp, Salmonella spp, Rhodococcus spp, Pseudomonasspp, Lactobacillus spp, Enterococcus spp, Alcaligenes spp, Klebsiellaspp, Paenibacillus spp, Arthrobacter spp, Corynebacterium spp,Brevibacterium spp, Thermus spp, Pseudomonas spp, Clostridium spp, andEscherichia spp. Non-limiting examples of bacteria that may be targetedby the methods and compositions provided herein are shown in Table 1. Insome instances, the 16S rRNA sequence of the bacteria targeted by themodulating agent has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%,99%, 99.9%, or 100% identity with a sequence listed in Table 1.

TABLE 1 Examples of Target Bacteria and Host InsectsPrimary endosymbiont Host Location 16S rRNA Gamma proteobacteriaCarsonella ruddii Psyllids bacteriocytes TATCCAGCCACAGGTTCCCCTAC(Psylloidea) AGCTACCTTGTTACGACTTCACC CCAGTTACAAATCATACCGTTGTAATAGTAAAATTACTTATGATACA ATTTACTTCCATGGTGTGACGGG CGGTGTGTACAAGGCTCGAGAACGTATTCACCGTAACATTCTGAT TTACGATTACTAGCGATTCCAAC TTCATGAAATCGAGTTACAGATTTCAATCCGAACTAAGAATATTTTT TAAGATTAGCATTATGTTGCCATATAGCATATAACTTTTTGTAATAC TCATTGTAGCACGTGTGTAGCCC TACTTATAAGGGCCATGATGACTTGACGTCGTCCTCACCTTCCTCC AATTTATCATTGGCAGTTTCTTATTAGTTCTAATATATTTTTAGTAAA ATAAGATAAGGGTTGCGCTCGTT ATAGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCA TGCAGCACCTGTCTCAAAGCTAA AAAAGCTTTATTATTTCTAATAAATTCTTTGGATGTCAAAAGTAGGT AAGATTTTTCGTGTTGTATCGAA TTAAACCACATGCTCCACCGCTTGTGCGAGCCCCCGTCAATTCAT TTGAGTTTTAACCTTGCGGTCGT AATCCCCAGGCGGTCAACTTAACGCGTTAGCTTTTTCACTAAAAA TATATAACTTTTTTTCATAAAACAAAATTACAATTATAATATTTAATA AATAGTTGACATCGTTTACTGCA TGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCATGCTTTCGTG TATTAGTGTCAGTATTAAAATAG AAATACGCCTTCGCCACTAGTATTCTTTCAGATATCTAAGCATTTCA CTGCTACTCCTGAAATTCTAATTTCTTCTTTTATACTCAAGTTTATA AGTATTAATTTCAATATTAAATTACTTTAATAAATTTAAAAATTAATT TTTAAAAACAACCTGCACACCCT TTACGCCCAATAATTCCGATTAACGCTTGCACCCCTCGTATTACC GCGGCTGCTGGCACGAAGTTAG CCGGTGCTTCTTTTACAAATAACGTCAAAGATAATATTTTTTTATTA TAAAATCTCTTCTTACTTTGTTGAAAGTGTTTTACAACCCTAAGGCC TTCTTCACACACGCGATATAGCT GGATCAAGCTTTCGCTCATTGTCCAATATCCCCCACTGCTGCCTTC CGTAAAAGTTTGGGCCGTGTCT CAGTCCCAATGTGGTTGTTCATCCTCTAAGATCAACTACGAATCAT AGTCTTGTTAAGCTTTTACTTTAA CAACTAACTAATTCGATATAAGCTCTTCTATTAGCGAACGACATTC TCGTTCTTTATCCATTAGGATAC ATATTGAATTACTATACATTTCTATATACTTTTCTAATACTAATAGGT AGATTCTTATATATTACTCACCCGTTCGCTGCTAATTATTTTTTTAA TAATTCGCACAACTTGCATGTGT TAAGCTTATCGCTAGCGTTCAATCTGAGCTATGATCAAACTCA (SEQ ID NO: 1) Portiera aleyrodidarum whiteflyesbacteriocytes AAGAGTTTGATCATGGCTCAGAT BT-B (Aleyrodoidea)TGAACGCTAGCGGCAGACATAA CACATGCAAGTCGAGCGGCATC ATACAGGTTGGCAAGCGGCGCACGGGTGAGTAATACATGTAAATA TACCTAAAAGTGGGGAATAACGT ACGGAAACGTACGCTAATACCGCATAATTATTACGAGATAAAGCA GGGGCTTGATAAAAAAAATCAAC CTTGCGCTTTTAGAAAATTACATGCCGGATTAGCTAGTTGGTAGA GTAAAAGCCTACCAAGGTAACG ATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGA GAAAAGGCCCAGACTCCTACGG GAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAACCCTGATC CAGTCATGCCGCGTGTGTGAAG AAGGCCTTTGGGTTGTAAAGCACTTTCAGCGAAGAAGAAAAGTTA GAAAATAAAAAGTTATAACTATG ACGGTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGC CGCGGTAAGACGGAGGGTGCAA GCGTTAATCAGAATTACTGGGCGTAAAGGGCATGTAGGTGGTTT GTTAAGCTTTATGTGAAAGCCCT ATGCTTAACATAGGAACGGAATAAAGAACTGACAAACTAGAGTGCA GAAGAGGAAGGTAGAATTCCCG GTGTAGCGGTGAAATGCGTAGATATCTGGAGGAATACCAGTTGC GAAGGCGACCTTCTGGGCTGAC ACTGACACTGAGATGCGAAAGCGTGGGGAGCAAACAGGATTAGA TACCCTGGTAGTCCACGCTGTAA ACGATATCAACTAGCCGTTGGATTCTTAAAGAATTTTGTGGCGTAG CTAACGCGATAAGTTGATCGCCT GGGGAGTACGGTCGCAAGGCTAAAACTCAAATGAATTGACGGGG GCCCGCACAAGCGGTGGAGCAT GTGGTTTAATTCGATGCAACGCGCAAAACCTTACCTACTCTTGACA TCCAAAGTACTTTCCAGAGATGG AAGGGTGCCTTAGGGAACTTTGAGACAGGTGCTGCATGGCTGTC GTCAGCTCGTGTTGTGAAATGTT GGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTGCCAA CGCATAAGGCGGGAACTTTAAG GAGACTGCTGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAA GTCATCATGGCCCTTAAGAGTAG GGCAACACACGTGCTACAATGGCAAAAACAAAGGGTCGCAAAAT GGTAACATGAAGCTAATCCCAAA AAAATTGTCTTAGTTCGGATTGGAGTCTGAAACTCGACTCCATAAA GTCGGAATCGCTAGTAATCGTG AATCAGAATGTCACGGTGAATACGTTCTCGGGCCTTGTACACACC GCCCGTCACACCATGGAAGTGA AATGCACCAGAAGTGGCAAGTTTAACCAAAAAACAGGAGAACAGT CACTACGGTGTGGTTCATGACT GGGGTGAAGTCGTAACAAGGTAGCTGTAGGGGAACCTGTGGCTG GATCACCTCCTTAA (SEQ ID NO: 2)Buchnera aphidicola str. Aphids bacteriocytes AGAGTTTGATCATGGCTCAGATTAPS (Acyrthosiphon (Aphidoidea) GAACGCTGGCGGCAAGCCTAAC pisum)ACATGCAAGTCGAGCGGCAGCG AGAAGAGAGCTTGCTCTCTTTGT CGGCAAGCGGCAAACGGGTGAGTAATATCTGGGGATCTACCCAA AAGAGGGGGATAACTACTAGAA ATGGTAGCTAATACCGCATAATGTTGAAAAACCAAAGTGGGGGAC CTTTTGGCCTCATGCTTTTGGAT GAACCCAGACGAGATTAGCTTGTTGGTAGAGTAATAGCCTACCAA GGCAACGATCTCTAGCTGGTCT GAGAGGATAACCAGCCACACTGGAACTGAGACACGGTCCAGACT CCTACGGGAGGCAGCAGTGGG GAATATTGCACAATGGGCGAAAGCCTGATGCAGCTATGCCGCGT GTATGAAGAAGGCCTTAGGGTT GTAAAGTACTTTCAGCGGGGAGGAAAAAAATAAAACTAATAATTTT ATTTCGTGACGTTACCCGCAGAA GAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAG GGTGCAAGCGTTAATCAGAATTA CTGGGCGTAAAGAGCGCGTAGGTGGTTTTTTAAGTCAGGTGTGAA ATCCCTAGGCTCAACCTAGGAA CTGCATTTGAAACTGGAAAACTAGAGTTTCGTAGAGGGAGGTAGA ATTCTAGGTGTAGCGGTGAAATG CGTAGATATCTGGAGGAATACCCGTGGCGAAAGCGGCCTCCTAA ACGAAAACTGACACTGAGGCGC GAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATG CCGTAAACGATGTCGACTTGGA GGTTGTTTCCAAGAGAAGTGACTTCCGAAGCTAACGCATTAAGTCG ACCGCCTGGGGAGTACGGCCG CAAGGCTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGG TGGAGCATGTGGTTTAATTCGAT GCAACGCGAAAAACCTTACCTGGTCTTGACATCCACAGAATTCTT TAGAAATAAAGAAGTGCCTTCGG GAGCTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTG TGAAATGTTGGGTTAAGTCCCGC AACGAGCGCAACCCTTATCCCCTGTTGCCAGCGGTTCGGCCGGG AACTCAGAGGAGACTGCCGGTT ATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCC TTACGACCAGGGCTACACACGT GCTACAATGGTTTATACAAAGAGAAGCAAATCTGCAAAGACAAGCA AACCTCATAAAGTAAATCGTAGT CCGGACTGGAGTCTGCAACTCGACTCCACGAAGTCGGAATCGCT AGTAATCGTGGATCAGAATGCCA CGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC ATGGGAGTGGGTTGCAAAAGAA GCAGGTATCCTAACCCTTTAAAAGGAAGGCGCTTACCACTTTGTG ATTCATGACTGGGGTGAAGTCG TAACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCCTT (SEQ ID NO: 3) Buchnera aphidicola str. Aphidsbacteriocytes AAACTGAAGAGTTTGATCATGGC Sg (Schizaphis (Aphidoidea)TCAGATTGAACGCTGGCGGCAA graminum) GCCTAACACATGCAAGTCGAGCGGCAGCGAAAAGAAAGCTTGCT TTCTTGTCGGCGAGCGGCAAAC GGGTGAGTAATATCTGGGGATCTGCCCAAAAGAGGGGGATAACT ACTAGAAATGGTAGCTAATACCG CATAAAGTTGAAAAACCAAAGTGGGGGACCTTTTTTAAAGGCCTCA TGCTTTTGGATGAACCCAGACGA GATTAGCTTGTTGGTAAGGTAAAAGCTTACCAAGGCAACGATCTCT AGCTGGTCTGAGAGGATAACCA GCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCA GCAGTGGGGAATATTGCACAAT GGGCGAAAGCCTGATGCAGCTATGCCGCGTGTATGAAGAAGGCC TTAGGGTTGTAAAGTACTTTCAG CGGGGAGGAAAAAATTAAAACTAATAATTTTATTTTGTGACGTTACC CGCAGAAGAAGCACCGGCTAAC TCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAAT CAGAATTACTGGGCGTAAAGAG CACGTAGGTGGTTTTTTAAGTCAGATGTGAAATCCCTAGGCTTAAC CTAGGAACTGCATTTGAAACTGA AATGCTAGAGTATCGTAGAGGGAGGTAGAATTCTAGGTGTAGCG GTGAAATGCGTAGATATCTGGA GGAATACCCGTGGCGAAAGCGGCCTCCTAAACGAATACTGACACT GAGGTGCGAAAGCGTGGGGAG CAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGTC GACTTGGAGGTTGTTTCCAAGA GAAGTGACTTCCGAAGCTAACGCGTTAAGTCGACCGCCTGGGGA GTACGGCCGCAAGGCTAAAACT CAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT TTAATTCGATGCAACGCGAAAAA CCTTACCTGGTCTTGACATCCACAGAATTTTTTAGAAATAAAAAAGT GCCTTCGGGAACTGTGAGACAG GTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTA AGTCCCGCAACGAGCGCAACCC TTATCCCCTGTTGCCAGCGGTTCGGCCGGGAACTCAGAGGAGACT GCCGGTTATAAACCGGAGGAAG GTGGGGACGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTA CACACGTGCTACAATGGTTTATA CAAAGAGAAGCAAATCTGTAAAGACAAGCAAACCTCATAAAGTAAA TCGTAGTCCGGACTGGAGTCTG CAACTCGACTCCACGAAGTCGGAATCGCTAGTAATCGTGGATCAG AATGCCACGGTGAATACGTTCC CGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGC AAAAGAAGCAGATTTCCTAACCA CGAAAGTGGAAGGCGTCTACCACTTTGTGATTCATGACTGGGGTG AAGTCGTAACAAGGTAACCGTA GGGGAACCTGCGGTTGGATCACCTCCTTA (SEQ ID NO: 4) Buchnera aphidicola str. Aphids bacteriocytesACTTAAAATTGAAGAGTTTGATC Bp (Baizongia pistaciae) (Aphidoidea)ATGGCTCAGATTGAACGCTGGC GGCAAGCTTAACACATGCAAGT CGAGCGGCATCGAAGAAAAGTTTACTTTTCTGGCGGCGAGCGGC AAACGGGTGAGTAACATCTGGG GATCTACCTAAAAGAGGGGGACAACCATTGGAAACGATGGCTAAT ACCGCATAATGTTTTTAAATAAA CCAAAGTAGGGGACTAAAATTTTTAGCCTTATGCTTTTAGATGAAC CCAGACGAGATTAGCTTGATGG TAAGGTAATGGCTTACCAAGGCGACGATCTCTAGCTGGTCTGAG AGGATAACCAGCCACACTGGAA CTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAAT ATTGCACAATGGGCTAAAGCCT GATGCAGCTATGCCGCGTGTATGAAGAAGGCCTTAGGGTTGTAA AGTACTTTCAGCGGGGAGGAAA GAATTATGTCTAATATACATATTTTGTGACGTTACCCGAAGAAGAA GCACCGGCTAACTCCGTGCCAG CAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCAGAATTACTG GGCGTAAAGAGCACGTAGGCGG TTTATTAAGTCAGATGTGAAATCCCTAGGCTTAACTTAGGAACTGC ATTTGAAACTAATAGACTAGAGT CTCATAGAGGGAGGTAGAATTCTAGGTGTAGCGGTGAAATGCGTA GATATCTAGAGGAATACCCGTG GCGAAAGCGACCTCCTAAATGAAAACTGACGCTGAGGTGCGAAA GCGTGGGGAGCAAACAGGATTA GATACCCTGGTAGTCCATGCTGTAAACGATGTCGACTTGGAGGTT GTTTCCTAGAGAAGTGGCTTCC GAAGCTAACGCATTAAGTCGACCGCCTGGGGAGTACGGTCGCAA GGCTAAAACTCAAATGAATTGAC GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCA ACGCGAAGAACCTTACCTGGTC TTGACATCCATAGAATTTTTTAGAGATAAAAGAGTGCCTTAGGGAA CTATGAGACAGGTGCTGCATGG CTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCCTATCCTTTGTT GCCATCAGGTTATGCTGGGAACTCAGAGGAGACTGCCGGTTATA AACCGGAGGAAGGTGGGGATGA CGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTA CAATGGCATATACAAAGAGATGC AACTCTGCGAAGATAAGCAAACCTCATAAAGTATGTCGTAGTCCGG ACTGGAGTCTGCAACTCGACTC CACGAAGTAGGAATCGCTAGTAATCGTGGATCAGAATGCCACGG TGAATACGTTCCCGGGCCTTGTA CACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGCAG GTAGCTTAACCAGATTATTTTATT GGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCG TAACAAGGTAACCGTAGGGGAA CCTGCGGTTGGATCACCTCCTTA(SEQ ID NO: 5) Buchnera aphidicola BCc Aphids bacteriocytesATGAGATCATTAATATATAAAAAT (Aphidoidea) CATGTTCCAATTAAAAAATTAGGACAAAATTTTTTACAGAATAAAGA AATTATTAATCAGATAATTAATTTAATAAATATTAATAAAAATGATAA TATTATTGAAATAGGATCAGGAT TAGGAGCGTTAACTTTTCCTATTTGTAGAATCATTAAAAAAATGAT AGTATTAGAAATTGATGAAGATC TTGTGTTTTTTTTAACTCAAAGTTTATTTATTAAAAAATTACAAATTA TAATTGCTGATATTATAAAATTTGATTTTTGTTGTTTTTTTTCTTTAC AGAAATATAAAAAATATAGGTTTATTGGTAATTTACCATATAATATTG CTACTATATTTTTTTTAAAAACAATTAAATTTCTTTATAATATAATTG ATATGCATTTTATGTTTCAAAAAGAAGTAGCAAAGAGATTATTAGCT ACTCCTGGTACTAAAGAATATGG TAGATTAAGTATTATTGCACAATATTTTTATAAGATAGAAACTGTTAT TAATGTTAATAAATTTAATTTTTTTCCTACTCCTAAAGTAGATTCTAC TTTTTTACGATTTACTCCTAAATATTTTAATAGTAAATATAAAATAGA TAAACATTTTTCTGTTTTAGAATTAATTACTAGATTTTCTTTTCAACA TAGAAGAAAATTTTTAAATAATAATTTAATATCTTTATTTTCTACAAA AGAATTAATTTCTTTAGATATTGATCCATATTCAAGAGCAGAAAATG TTTCTTTAATTCAATATTGTAAATTAATGAAATATTATTTGAAAAGAA AAATTTTATGTTTAGATTAA (SEQ ID NO: 6)Buchnera aphidicola Aphids bacteriocytes TTATCTTATTTCACATATACGTAA(Cinara tujafilina) (Aphidoidea) TATTGCGCTGCGTGCACGAGGATTTTTTTGAATTTCAGATATATTT GGTTTAATACGTTTAATAAAACGTATTTTTTTTTTTATTTTTCTTATT TGCAATTCAGTAATAGGAAGTTTTTTAGGTATATTTGGATAATTACT GTAATTCTTAATAAAGTTTTTTACAATCCTATCTTCAATAGAATGAA AACTAATAATAGCAATTTTTGATC CGGAATGTAATATGTTAATAATAATTTTTAATATTTTATGTAATTCA TTTATTTCTTGGTTAATATATATTCGAAAAGCTTGAAATGTTCTCGT AGCTGGATGTTTAAATTTGTCAT ATTTTGGGATTGATTTTTTTATGATTTGAACTAACTCTAACGTGCTT GTTATGGTTTTTTTTTTTATTTGT AATATGATGGCTCGGGATATTTTTTTTGCGTATTTTTCTTCGCCAAA ATTTTTTATTACCTGTTCTATTGTTTTTTGGTTTGTTTTTTTTAACCA TTGACTAACTGATATTCCAGATT TAGGGTTCATACGCATATCTAAAGGTCCATCATTCATAAATGAAAA TCCTCGGATACTAGAATTTAACT GTATTGAAGAAATACCTAAATCTAATAATATTCCATCTATTTTATCT CTATTTTTTTCTTTTTTTAATATTTTTTCAATATTAGAAAATTTACCTA AAAATATTTTAAATCGCGAATCTTTTATTTTTTTTCCGATTTTTATAG ATTGTGGGTCTTGATCAATACTATATAACTTTCCATTAACCCCTAAT TCTTGAAGAATTGCTTTTGAATG ACCACCACCTCCAAATGTACAATCAACATATGTACCGTCTTTTTTTA TTTTTAAGTATTGTATGATTTCTTTTGTTAAAACAGGTTTATGAATC AT (SEQ ID NO: 7) Buchnera aphidicola str.Aphids bacteriocytes ATGAAAAGTATAAAAACTTTTAAA G002 (Myzus persicae)(Aphidoidea) AAACACTTTCCTGTGAAAAAATA TGGACAAAATTTTCTTATTAATAAAGAGATCATAAAAAATATTGTTA AAAAAATTAATCCAAATATAGAA CAAACATTAGTAGAAATCGGACCAGGATTAGCTGCATTAACTGAGC CCATATCTCAGTTATTAAAAGAG TTAATAGTTATTGAAATAGACTGTAATCTATTATATTTTTTAAAAAAA CAACCATTTTATTCAAAATTAATAGTTTTTTGTCAAGATGCTTTAAA CTTTAATTATACAAATTTATTTTATAAAAAAAATAAATTAATTCGTAT TTTTGGTAATTTACCATATAATATCTCTACATCTTTAATTATTTTTTT ATTTCAACACATTAGAGTAATTCAAGATATGAATTTTATGCTTCAAA AAGAAGTTGCTGCAAGATTAATT GCATTACCTGGAAATAAATATTACGGTCGTTTGAGCATTATATCTC AATATTATTGTGATATCAAAATTT TATTAAATGTTGCTCCTGAAGATTTTTGGCCTATTCCGAGAGTTCA TTCTATATTTGTAAATTTAACACCTCATCATAATTCTCCTTATTTTGT TTATGATATTAATATTTTAAGCOTTATTACAAATAAGGCTTTCCAAA ATAGAAGAAAAATATTACGTCAT AGTTTAAAAAATTTATTTTCTGAAACAACTTTATTAAATTTAGATATT AATCCCAGATTAAGAGCTGAAAATATTTCTGTTTTTCAGTATTGTCA ATTAGCTAATTATTTGTATAAAAA AAATTATACTAAAAAAAATTAA(SEQ ID NO: 8) Buchnera aphidicola str. Aphids bacteriocytesATTATAAAAAATTTTAAAAAACAT Ak (Acyrthosiphon (Aphidoidea)TTTCCTTTAAAAAGGTATGGACA kondoi) AAATTTTCTTGTCAATACAAAAACTATTCAAAAGATAATTAATATAAT TAATCCAAACACCAAACAAACAT TAGTGGAAATTGGACCTGGATTAGCTGCATTAACAAAACCAATTTG TCAATTATTAGAAGAATTAATTGTTATTGAAATAGATCCTAATTTATT GTTTTTATTAAAAAAACGTTCATTTTATTCAAAATTAACAGTTTTTTA TCAAGACGCTTTAAATTTCAATTATACAGATTTGTTTTATAAGAAAAA TCAATTAATTCGTGTTTTTGGAAACTTGCCATATAATATTTCTACATC TTTAATTATTTCTTTATTCAATCATATTAAAGTTATTCAAGATATGAA TTTTATGTTACAGAAAGAGGTTG CTGAAAGATTAATTTCTATTCCTGGAAATAAATCTTATGGCCGTTT AAGCATTATTTCTCAGTATTATTGTAAAATTAAAATATTATTAAATGT TGTACCTGAAGATTTTCGACCTA TACCGAAAGTGCATTCTGTTTTTATCAATTTAACTCCTCATACCAAT TCTCCATATTTTGTTTATGATACAAATATCCTCAGTTCTATCACAAG AAATGCTTTTCAAAATAGAAGGA AAATTTTGCGTCATAGTTTAAAAAATTTATTTTCTGAAAAAGAACTAA TTCAATTAGAAATTAATCCAAATTTACGAGCTGAAAATATTTCTATC TTTCAGTATTGTCAATTAGCTGA TTATTTATATAAAAAATTAAATAATCTTGTAAAAATCAATTAA (SEQ ID NO: 9) Buchnera aphidicola str. Aphidsbacteriocytes ATGATACTAAATAAATATAAAAAA Ua (Uroleucon (Aphidoidea)TTTATTCCTTTAAAAAGATACGG ambrosiae) ACAAAATTTTCTTGTAAATAGAGAAATAATCAAAAATATTATCAAAA TAATTAATCCTAAAAAAACGCAA ACATTATTAGAAATTGGACCGGGTTTAGGTGCGTTAACAAAACCTA TTTGTGAATTTTTAAATGAACTTA TCGTCATTGAAATAGATCCTAATATATTATCTTTTTTAAAGAAATGT ATATTTTTTGATAAATTAAAAATATATTGTCATAATGCTTTAGATTTT AATTATAAAAATATATTCTATAAAAAAAGTCAATTAATTCGTATTTTT GGAAATTTACCATATAATATTTCTACATCTTTAATAATATATTTATTT CGGAATATTGATATTATTCAAGATATGAATTTTATGTTACAACAAGA AGTGGCTAAAAGATTAGTTGCTA TTCCTGGTGAAAAACTTTATGGTCGTTTAAGTATTATATCTCAATAT TATTGTAATATTAAAATATTATTACATATTCGACCTGAAAATTTTCA ACCTATTCCTAAAGTTAATTCAAT GTTTGTAAATTTAACTCCGCATATTCATTCTCCTTATTTTGTTTATG ATATTAATTTATTAACTAGTATTACAAAACATGCTTTTCAACATAGA AGAAAAATATTGCGTCATAGTTT AAGAAATTTTTTTTCTGAGCAAGATTTAATTCATTTAGAAATTAATC CAAATTTAAGAGCTGAAAATGTTTCTATTATTCAATATTGTCAATTG GCTAATAATTTATATAAAAAACAT AAACAGTTTATTAATAATTAA(SEQ ID NO: 10) Buchnera aphidicola Aphids bacteriocytesATGAAAAAGCATATTCCTATAAA (Aphis glycines) (Aphidoidea)AAAATTTAGTCAAAATTTTCTTGT AGATTTGAGTGTGATTAAAAAAATAATTAAATTTATTAATCCGCAGT TAAATGAAATATTGGTTGAAATT GGACCGGGATTAGCTGCTATCACTCGACCTATTTGTGATTTGATA GATCATTTAATTGTGATTGAAATTGATAAAATTTTATTAGATAGATTA AAACAGTTCTCATTTTATTCAAAATTAACAGTATATCATCAAGATGC TTTAGCATTTGATTACATAAAGTTATTTAATAAAAAAAATAAATTAGT TCGAATTTTTGGTAATTTACCATATCATGTTTCTACGTCTTTAATATT GCATTTATTTAAAAGAATTAATATTATTAAAGATATGAATTTTATGCT ACAAAAAGAAGTTGCTGAACGTT TAATTGCAACTCCAGGTAGTAAATTATATGGTCGTTTAAGTATTATT TCTCAATATTATTGTAATATAAAAGTTTTATTGCATGTGTCTTCAAA ATGTTTTAAACCAGTTCCTAAAG TAGAATCAATTTTTCTTAATTTGACACCTTATACTGATTATTTCCCTT ATTTTACTTATAATGTAAACGTTCTTAGTTATATTACAAATTTAGCTT TTCAAAAAAGAAGAAAAATATTA CGTCATAGTTTAGGTAAAATATTTTCTGAAAAAGTTTTTATAAAATT AAATATTAATCCCAAATTAAGACCTGAGAATATTTCTATATTACAAT ATTGTCAGTTATCTAATTATATGATAGAAAATAATATTCATCAGGAA CATGTTTGTATTTAA (SEQ ID NO: 11)Annandia pinicola (Phylloxeroidea) bacteriocytes AGATTGAACGCTGGCGGCATGCCTTACACATGCAAGTCGAACGGT AACAGGTCTTCGGACGCTGACG AGTGGCGAACGGGTGAGTAATACATCGGAACGTGCCCAGTCGTG GGGGATAACTACTCGAAAGAGT AGCTAATACCGCATACGATCTGAGGATGAAAGCGGGGGACCTTCG GGCCTCGCGCGATTGGAGCGG CCGATGGCAGATTAGGTAGTTGGTGGGATAAAAGCTTACCAAGC CGACGATCTGTAGCTGGTCTGA GAGGACGACCAGCCACACTGGAACTGAGATACGGTCCAGACTCTT ACGGGAGGCAGCAGTGGGGAAT ATTGCACAATGGGCGCAAGCCTGATGCAGCTATGTCGCGTGTAT GAAGAAGACCTTAGGGTTGTAAA GTACTTTCGATAGCATAAGAAGATAATGAGACTAATAATTTTATTGT CTGACGTTAGCTATAGAAGAAGC ACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGGGGGTG CTAGCGTTAATCGGAATTACTGG GCGTAAAGAGCATGTAGGTGGTTTATTAAGTCAGATGTGAAATCC CTGGACTTAATCTAGGAACTGCA TTTGAAACTAATAGGCTAGAGTTTCGTAGAGGGAGGTAGAATTCT AGGTGTAGCGGTGAAATGCATA GATATCTAGAGGAATATCAGTGGCGAAGGCGACCTTCTGGACGAT AACTGACGCTAAAATGCGAAAG CATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATGCTGTA AACGATGTCGACTAAGAGGTTG GAGGTATAACTTTTAATCTCTGTAGCTAACGCGTTAAGTCGACCG CCTGGGGAGTACGGTCGCAAGG CTAAAACTCAAATGAATTGACGGGGGCCTGCACAAGCGGTGGAG CATGTGGTTTAATTCGATGCAAC GCGTAAAACCTTACCTGGTCTTGACATCCACAGAATTTTACAGAAA TGTAGAAGTGCAATTTGAACTGT GAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGT TGGGTTAAGTCCCGCAACGAGC GCAACCCTTGTCCTTTGTTACCATAAGATTTAAGGAACTCAAAGGA GACTGCCGGTGATAAACTGGAG GAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTATGACCAGG GCTACACACGTGCTACAATGGC ATATACAAAGAGATGCAATATTGCGAAATAAAGCCAATCTTATAAA ATATGTCCTAGTTCGGACTGGAG TCTGCAACTCGACTCCACGAAGTCGGAATCGCTAGTAATCGTGGA TCAGCATGCCACGGTGAATATGT TTCCAGGCCTTGTACACACCGCCCGTCACACCATGGAAGTGGAT TGCAAAAGAAGTAAGAAAATTAA CCTTCTTAACAAGGAAATAACTTACCACTTTGTGACTCATAACTGG GGTGA (SEQ ID NO: 12) Moranella endobia(Coccoidea) bacteriocytes TCTTTTTGGTAAGGAGGTGATCC AACCGCAGGTTCCCCTACGGTTACCTTGTTACGACTTCACCCCAG TCATGAATCACAAAGTGGTAAGC GCCCTCCTAAAAGGTTAGGCTACCTACTTCTTTTGCAACCCACTT CCATGGTGTGACGGGCGGTGTG TACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGAT TACTAGCGATTCCTACTTCATGG AGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGA GGTCCGCTAACTCTCGCGAGCT TGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTACT CGTAAGGGCCATGATGACTTGA CGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCTTTG AGTTCCCGACCGAATCGCTGGC AAAAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACA TTTCACAACACGAGCTGACGACA GCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGTACCAAAACATC TCTGCTAAGTTCTCTGGATGTCA AGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGC TCCACCGCTTGTGCGGGCCCCC GTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGG TCGATTTAACGCGTTAACTACGA AAGCCACAGTTCAAGACCACAGCTTTCAAATCGACATAGTTTACG GCGTGGACTACCAGGGTATCTA ATCCTGTTTGCTCCCCACGCTTTCGTACCTGAGCGTCAGTATTCGT CCAGGGGGCCGCCTTCGCCACT GGTATTCCTCCAGATATCTACACATTTCACCGCTACACCTGGAATT CTACCCCCCTCTACGAGACTCTA GCCTATCAGTTTCAAATGCAGTTCCTAGGTTAAGCCCAGGGATTT CACATCTGACTTAATAAACCGCC TACGTACTCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTC CGTATTACCGCGGCTGCTGGCA CGGAGTTAGCCGGTGCTTCTTCTGTAGGTAACGTCAATCAATAAC CGTATTAAGGATATTGCCTTCCT CCCTACTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACG CGGCATGGCTGCATCAGGGTTT CCCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCT GGACCGTGTCTCAGTTCCAGTG TGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGT AAGCTATTACCTCACCTACTAGC TAATCCCATCTGGGTTCATCTGAAGGTGTGAGGCCAAAAGGTCCC CCACTTTGGTCTTACGACATTAT GCGGTATTAGCTACCGTTTCCAGCAGTTATCCCCCTCCATCAGGCA GATCCCCAGACTTTACTCACCCG TTCGCTGCTCGCCGGCAAAAAAGTAAACTTTTTTCCGTTGCCGCT CAACTTGCATGTGTTAGGCCTGC CGCCAGCGTTCAATCTGAGCCATGATCAAACTCTTCAATTAAA (SEQ ID NO: 13) Ishikawaella capsulata(Heteroptera) bacteriocytes AAATTGAAGAGTTTGATCATGGC MpkobeTCAGATTGAACGCTAGCGGCAA GCTTAACACATGCAAGTCGAAC GGTAACAGAAAAAAGCTTGCTTTTTTGCTGACGAGTGGCGGACGG GTGAGTAATGTCTGGGGATCTA CCTAATGGCGGGGGATAACTACTGGAAACGGTAGCTAATACCGC ATAATGTTGTAAAACCAAAGTGG GGGACCTTATGGCCTCACACCATTAGATGAACCTAGATGGGATTA GCTTGTAGGTGGGGTAAAGGCT CACCTAGGCAACGATCCCTAGCTGGTCTGAGAGGATGACCAGCC ACACTGGAACTGAGATACGGTC CAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTGCACAATGGG CGCAAGCCTGATGCAGCTATGT CGCGTGTATGAAGAAGGCCTTAGGGTTGTAAAGTACTTTCATCGG GGAAGAAGGATATGAGCCTAAT ATTCTCATATATTGACGTTACCTGCAGAAGAAGCACCGGCTAACT CCGTGCCAGCAGCCGCGGTAAC ACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGAGC ACGTAGGTGGTTTATTAAGTCAT ATGTGAAATCCCTGGGCTTAACCTAGGAACTGCATGTGAAACTGAT AAACTAGAGTTTCGTAGAGGGA GGTGGAATTCCAGGTGTAGCGGTGAAATGCGTAGATATCTGGAG GAATATCAGAGGCGAAGGCGAC CTTCTGGACGAAAACTGACACTCAGGTGCGAAAGCGTGGGGAGCA AACAGGATTAGATACCCTGGTAG TCCACGCTGTAAACAATGTCGACTAAAAAACTGTGAGCTTGACTTG TGGTTTTTGTAGCTAACGCATTA AGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATG AATTGACGGGGGTCCGCACAAG CGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTAC CTGGTCTTGACATCCAGCGAATT ATATAGAAATATATAAGTGCCTTTCGGGGAACTCTGAGACGCTGCA TGGCTGTCGTCAGCTCGTGTTG TGAAATGTTGGGTTAAGTCCCGCAACGAGCGCCCTTATCCTCTGTT GCCAGCGGCATGGCCGGGAACT CAGAGGAGACTGCCAGTATTAAACTGGAGGAAGGTGGGGATGAC GTCAAGTCATCATGGCCCTTATG ACCAGGGCTACACACGTGCTACAATGGTGTATACAAAGAGAAGCA ATCTCGCAAGAGTAAGCAAAACT CAAAAAGTACATCGTAGTTCGGATTAGAGTCTGCAACTCGACTCTA TGAAGTAGGAATCGCTAGTAATC GTGGATCAGAATGCCACGGTGAATACGTTCTCTGGCCTTGTACAC ACCGCCCGTCACACCATGGGAG TAAGTTGCAAAAGAAGTAGGTAGCTTAACCTTTATAGGAGGGCGCT TACCACTTTGTGATTTATGACTG GGGTGAAGTCGTAACAAGGTAACTGTAGGGGAACCTGTGGTTGG ATTACCTCCTTA (SEQ ID NO: 14) Baumanniasharpshooter bacteriocytes TTCAATTGAAGAGTTTGATCATG cicadellinicolaleafhoppers GCTCAGATTGAACGCTGGCGGT (Cicadellinae)AAGCTTAACACATGCAAGTCGAG CGGCATCGGAAAGTAAATTAATT ACTTTGCCGGCAAGCGGCGAACGGGTGAGTAATATCTGGGGATC TACCTTATGGAGAGGGATAACTA TTGGAAACGATAGCTAACACCGCATAATGTCGTCAGACCAAAATG GGGGACCTAATTTAGGCCTCAT GCCATAAGATGAACCCAGATGAGATTAGCTAGTAGGTGAGATAAT AGCTCACCTAGGCAACGATCTCT AGTTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACG GTCCAGACTCCTACGGGAGGCA GCAGTGGGGAATCTTGCACAATGGGGGAAACCCTGATGCAGCTA TACCGCGTGTGTGAAGAAGGCC TTCGGGTTGTAAAGCACTTTCAGCGGGGAAGAAAATGAAGTTACT AATAATAATTGTCAATTGACGTTA CCCGCAAAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGT AAGACGGAGGGTGCAAGCGTTA ATCGGAATTACTGGGCGTAAAGCGTATGTAGGCGGTTTATTTAGT CAGGTGTGAAAGCCCTAGGCTT AACCTAGGAATTGCATTTGAAACTGGTAAGCTAGAGTCTCGTAGA GGGGGGGAGAATTCCAGGTGTA GCGGTGAAATGCGTAGAGATCTGGAAGAATACCAGTGGCGAAGG CGCCCCCCTGGACGAAAACTGA CGCTCAAGTACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCT GGTAGTCCACGCTGTAAACGAT GTCGATTTGAAGGTTGTAGCCTTGAGCTATAGCTTTCGAAGCTAAC GCATTAAATCGACCGCCTGGGG AGTACGACCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCG CACAAGCGGTGGAGCATGTGGT TTAATTCGATACAACGCGAAAAACCTTACCTACTCTTGACATCCAG AGTATAAAGCAGAAAAGCTTTAG TGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAG CTCGTGTTGTGAAATGTTGGGTT AAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAACGATT AAGTCGGGAACTCAAAGGAGAC TGCCGGTGATAAACCGGAGGAAGGTGAGGATAACGTCAAGTCAT CATGGCCCTTACGAGTAGGGCT ACACACGTGCTACAATGGTGCATACAAAGAGAAGCAATCTCGTAAG AGTTAGCAAACCTCATAAAGTGC ATCGTAGTCCGGATTAGAGTCTGCAACTCGACTCTATGAAGTCGGA ATCGCTAGTAATCGTGGATCAGA ATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGT CACACCATGGGAGTGTATTGCA AAAGAAGTTAGTAGCTTAACTCATAATACGAGAGGGCGCTTACCA CTTTGTGATTCATAACTGGGGTG AAGTCGTAACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCAC CTCCTTACACTAAA (SEQ ID NO: 15) Sodalis likeRhopalus wider tissue ATTGAACGCTGGCGGCAGGCCT sapporensis tropismAACACATGCAAGTCGAGCGGCA GCGGGAAGAAGCTTGCTTCTTT GCCGGCGAGCGGCGGACGGGTGAGTAATGTCTGGGGATCTGCC CGATGGAGGGGGATAACTACTG GAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGG GACCTTCGGGCCTCACACCATC GGATGAACCCAGGTGGGATTAGCTAGTAGGTGGGGTAATGGCTC ACCTAGGCGACGATCCCTAGCT GGTCTGAGAGGATGACCAGTCACACTGGAACTGAGACACGGTCC AGACTCCTACGGGAGGCAGCAG TGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCCATGCC GCGTGTGTGAAGAAGGCCTTCG GGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGATGGCGTTAAT AGCGCTATCGATTGACGTTACCC GCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAAT ACGGAGGGTGCGAGCGTTAATC GGAATTACTGGGCGTAAAGCGTACGCAGGCGGTCTGTTAAGTCA GATGTGAAATCCCCGGGCTCAA CCTGGGAACTGCATTTGAAACTGGCAGGCTAGAGTCTCGTAGAGG GGGGTAGAATTCCAGGTGTAGC GGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCG GCCCCCTGGACGAAGACTGACG CTCAGGTACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTG GTAGTCCACGCTGTAAACGATGT CGATTTGAAGGTTGTGGCCTTGAGCCGTGGCTTTCGGAGCTAACG TGTTAAATCGACCGCCTGGGGA GTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCG CACAAGCGGTGGAGCATGTGGT TTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAG AGAACTTGGCAGAGATGCTTTG GTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCA GCTCGTGTTGTGAAATGTTGGGT TAAGTCCCGCAACGAGCGCAACCCTTATCCTTTATTGCCAGCGAT TCGGTCGGGAACTCAAAGGAGA CTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCA TCATGGCCCTTACGAGTAGGGC TACACACGTGCTACAATGGCGCATACAAAGAGAAGCGATCTCGC GAGAGTCAGCGGACCTCATAAA GTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAA GTCGGAATCGCTAGTAATCGTG GATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACAC CGCCCGTCACACCATGGGAGTG GGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTA CCACTTTGTGATTCATGACTGGG GTG (SEQ ID NO: 16)Hartigia pinicola The pine bark bacteriocytes AGATTTAACGCTGGCGGCAGGCadelgid CTAACACATGCAAGTCGAGCGG TACCAGAAGAAGCTTGCTTCTTGCTGACGAGCGGCGGACGGGTG AGTAATGTATGGGGATCTGCCC GACAGAGGGGGATAACTATTGGAAACGGTAGCTAATACCGCATAA TCTCTGAGGAGCAAAGCAGGGG AACTTCGGTCCTTGCGCTATCGGATGAACCCATATGGGATTAGCT AGTAGGTGAGGTAATGGCTCCC CTAGGCAACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACA CTGGGACTGAGACACGGCCCAG ACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGA AAGCCTGATGCAGCCATGCCGC GTGTATGAAGAAGGCTTTAGGGTTGTAAAGTACTTTCAGTCGAGA GGAAAACATTGATGCTAATATCA TCAATTATTGACGTTTCCGACAGAAGAAGCACCGGCTAACTCCGT GCCAGCAGCCGCGGTAATACGG AGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGC AGGCGGTTAATTAAGTTAGATGT GAAAGCCCCGGGCTTAACCCAGGAATAGCATATAAAACTGGTCAA CTAGAGTATTGTAGAGGGGGGT AGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAAT ACCAGTGGCGAAGGCGGCCCC CTGGACAAAAACTGACGCTCAAATGCGAAAGCGTGGGGAGCAAAC AGGATTAGATACCCTGGTAGTCC ATGCTGTAAACGATGTCGATTTGGAGGTTGTTCCCTTGAGGAGTA GCTTCCGTAGCTAACGCGTTAAA TCGACCGCCTGGGGGAGTACGACTGCAAGGTTAAAACTCAAATGA ATTGACGGGGGCCCGCACAAGC GGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTAC CTACTCTTGACATCCAGATAATT TAGCAGAAATGCTTTAGTACCTTCGGGAAATCTGAGACAGGTGCT GCATGGCTGTCGTCAGCTCGTG TTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATC CTTTGTTGCCAGCGATTAGGTCG GGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGG GATGACGTCAAGTCATCATGGC CCTTACGAGTAGGGCTACACACGTGCTACAATGGCATATACAAAG GGAAGCAACCTCGCGAGAGCAA GCGAAACTCATAAATTATGTCGTAGTTCAGATTGGAGTCTGCAACT CGACTCCATGAAGTCGGAATCG CTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGC CTTGTACACACCGCCCGTCACA CCATGGGAGTGGGTTGCAAAAGAAGTAGGTAACTTAACCTTATGG AAAGCGCTTACCACTTTGTGATT CATAACTGGGGTG(SEQ ID NO: 17) Wigglesworthia tsetse fly bacteriocytes glossinidia(Diptera: Glossinidae) Beta proteobacteria Tremblaya phenacolaPhenacoccus bacteriomes AGGTAATCCAGCCACACCTTCCA avenaeGTACGGCTACCTTGTTACGACTT (TPPAVE). CACCCCAGTCACAACCCTTACCTTCGGAACTGCCCTCCTCACAACT CAAACCACCAAACACTTTTAAAT CAGGTTGAGAGAGGTTAGGCCTGTTACTTCTGGCAAGAATTATTT CCATGGTGTGACGGGCGGTGTG TACAAGACCCGAGAACATATTCACCGTGGCATGCTGATCCACGAT TACTAGCAATTCCAACTTCATGC ACTCGAGTTTCAGAGTACAATCCGAACTGAGGCCGGCTTTGTGAG ATTAGCTCCCTTTTGCAAGTTGG CAACTCTTTGGTCCGGCCATTGTATGATGTGTGAAGCCCCACCCA TAAAGGCCATGAGGACTTGACG TCATCCCCACCTTCCTCCAACTTATCGCTGGCAGTCTCTTTAAGGT AACTGACTAATCCAGTAGCAATT AAAGACAGGGGTTGCGCTCGTTACAGGACTTAACCCAACATCTCA CGACACGAGCTGACGACAGCCA TGCAGCACCTGTGCACTAATTCTCTTTCAAGCACTCCCGCTTCTCA ACAGGATCTTAGCCATATCAAAG GTAGGTAAGGTTTTTCGCGTTGCATCGAATTAATCCACATCATCCA CTGCTTGTGCGGGTCCCCGTCA ATTCCTTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCG ACTTGTGCGTTAGCTGCACCACT GAAAAGGAAAACTGCCCAATGGTTAGTCAACATCGTTTAGGGCAT GGACTACCAGGGTATCTAATCCT GTTTGCTCCCCATGCTTTAGTGTCTGAGCGTCAGTAACGAACCAG GAGGCTGCCTACGCTTTCGGTA TTCCTCCACATCTCTACACATTTCACTGCTACATGCGGAATTCTAC CTCCCCCTCTCGTACTCCAGCCT GCCAGTAACTGCCGCATTCTGAGGTTAAGCCTCAGCCTTTCACAG CAATCTTAACAGGCAGCCTGCA CACCCTTTACGCCCAATAAATCTGATTAACGCTCGCACCCTACGTA TTACCGCGGCTGCTGGCACGTA GTTTGCCGGTGCTTATTCTTTCGGTACAGTCACACCACCAAATTGT TAGTTGGGTGGCTTTCTTTCCGA ACAAAAGTGCTTTACAACCCAAAGGCCTTCTTCACACACGCGGCA TTGCTGGATCAGGCTTCCGCCC ATTGTCCAAGATTCCTCACTGCTGCCTTCCTCAGAAGTCTGGGCC GTGTCTCAGTCCCAGTGTGGCT GGCCGTCCTCTCAGACCAGCTACCGATCATTGCCTTGGGAAGCC ATTACCTTTCCAACAAGCTAATC AGACATCAGCCAATCTCAGAGCGCAAGGCAATTGGTCCCCTGCT TTCATTCTGCTTGGTAGAGAACT TTATGCGGTATTAATTAGGCTTTCACCTAGCTGTCCCCCACTCTG AGGCATGTTCTGATGCATTACTC ACCCGTTTGCCACTTGCCACCAAGCCTAAGCCCGTGTTGCCGTTC GACTTGCATGTGTAAGGCATGC CGCTAGCGTTCAATCTGAGCCAGGATCAAACTCT (SEQ ID NO: 18) Tremblaya princeps citrus mealybugbacteriomes AGAGTTTGATCCTGGCTCAGATT Planococcus citriGAACGCTAGCGGCATGCATTAC ACATGCAAGTCGTACGGCAGCA CGGGCTTAGGCCTGGTGGCGAGTGGCGAACGGGTGAGTAACGCC TCGGAACGTGCCTTGTAGTGGG GGATAGCCTGGCGAAAGCCAGATTAATACCGCATGAAGCCGCACA GCATGCGCGGTGAAAGTGGGG GATTCTAGCCTCACGCTACTGGATCGGCCGGGGTCTGATTAGCTA GTTGGCGGGGTAATGGCCCACC AAGGCTTAGATCAGTAGCTGGTCTGAGAGGACGATCAGCCACAC TGGGACTGAGACACGGCCCAGA CTCCTACGGGAGGCAGCAGTGGGGAATCTTGGACAATGGGCGCA AGCCTGATCCAGCAATGCCGCG TGTGTGAAGAAGGCCTTCGGGTCGTAAAGCACTTTTGTTCGGGAT GAAGGGGGGCGTGCAAACACCA TGCCCTCTTGACGATACCGAAAGAATAAGCACCGGCTAACTACG TGCCAGCAGCCGCGGTAATACG TAGGGTGCGAGCGTTAATCGGAATCACTGGGCGTAAAGGGTGCG CGGGTGGTTTGCCAAGACCCCT GTAAAATCCTACGGCCCAACCGTAGTGCTGCGGAGGTTACTGGT AAGCTTGAGTATGGCAGAGGGG GGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGATATCTGGAG GAATACCGAAGGCGAAGGCAAC CCCCTGGGCCATCACTGACACTGAGGCACGAAAGCGTGGGGAG CAAACAGGATTAGATACCCTGGT AGTCCACGCCCTAAACCATGTCGACTAGTTGTCGGGGGGAGCCC TTTTTCCTCGGTGACGAAGCTAA CGCATGAAGTCGACCGCCTGGGGAGTACGACCGCAAGGTTAAAA CTCAAAGGAATTGACGGGGACC CGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAA AACCTTACCTACCCTTGACATGG CGGAGATTCTGCCGAGAGGCGGAAGTGCTCGAAAGAGAATCCGT GCACAGGTGCTGCATGGCTGTC GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCATAACGAGC GCAACCCCCGTCTTTAGTTGCTA CCACTGGGGCACTCTATAGAGACTGCCGGTGATAAACCGGAGGA AGGTGGGGACGACGTCAAGTCA TCATGGCCTTTATGGGTAGGGCTTCACACGTCATACAATGGCTGG AGCAAAGGGTCGCCAACTCGAG AGAGGGAGCTAATCCCACAAACCCAGCCCCAGTTCGGATTGCAC TCTGCAACTCGAGTGCATGAAGT CGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACG TTCTCGGGTCTTGTACACACCGC CCGTCACACCATGGGAGTAAGCCGCATCAGAAGCAGCCTCCCTA ACCCTATGCTGGGAAGGAGGCT GCGAAGGTGGGGTCTATGACTGGGGTGAAGTCGTAACAAGGTAG CCGTACCGGAAGGTGCGGCTGG ATTACCT (SEQ ID NO: 19)Vidania bacteriomes Nasuia deltocephalinicola pestiferous insectbacteriomes AGTTTAATCCTGGCTCAGATTTA host, MacrostelesACGCTTGCGACATGCCTAACAC quadripunctulatus ATGCAAGTTGAACGTTGAAAATA(Hemiptera: TTTCAAAGTAGCGTATAGGTGAG Cicadellidae)TATAACATTTAAACATACCTTAAA GTTCGGAATACCCCGATGAAAAT CGGTATAATACCGTATAAAAGTATTTAAGAATTAAAGCGGGGAAAA CCTCGTGCTATAAGATTGTTAAA TGCCTGATTAGTTTGTTGGTTTTTAAGGTAAAAGCTTACCAAGACT TTGATCAGTAGCTATTCTGTGAG GATGTATAGCCACATTGGGATTGAAATAATGCCCAAACCTCTACGG AGGGCAGCAGTGGGGAATATTG GACAATGAGCGAAAGCTTGATCCAGCAATGTCGCGTGTGCGATT AAGGGAAACTGTAAAGCACTTTT TTTTAAGAATAAGAAATTTTAATTAATAATTAAAATTTTTGAATGTAT TAAAAGAATAAGTACCGACTAAT CACGTGCCAGCAGTCGCGGTAATACGTGGGGTGCGAGCGTTAAT CGGATTTATTGGGCGTAAAGTGT ATTCAGGCTGCTTAAAAAGATTTATATTAAATATTTAAATTAAATTT AAAAAATGTATAAATTACTATTAAGCTAGAGTTTAGTATAAGAAAAA AGAATTTTATGTGTAGCAGTGAA ATGCGTTGATATATAAAGGAACGCCGAAAGCGAAAGCATTTTTCTG TAATAGAACTGACGCTTATATAC GAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACG CCCTAAACTATGTCAATTAACTA TTAGAATTTTTTTTAGTGGTGTAGCTAACGCGTTAAATTGACCGCCT GGGTATTACGATCGCAAGATTAA AACTCAAAGGAATTGACGGGGACCAGCACAAGCGGTGGATGATG TGGATTAATTCGATGATACGCGA AAAACCTTACCTGCCCTTGACATGGTTAGAATTTTATTGAAAAATAA AAGTGCTTGGAAAAGAGCTAACA CACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTT GGGTTAAGTCCCGCAACGAGCG CAACCCCTACTCTTAGTTGCTAATTAAAGAACTTTAAGAGAACAGC TAACAATAAGTTTAGAGGAAGGA GGGGATGACTTCAAGTCCTCATGGCCCTTATGGGCAGGGCTTCA CACGTCATACAATGGTTAATACA AAAAGTTGCAATATCGTAAGATTGAGCTAATCTTTAAAATTAATCTT AGTTCGGATTGTACTCTGCAACT CGAGTACATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATG CCGCGGTGAATAGTTTAACTGGT CTTGTACACACCGCCCGTCACACCATGGAAATAAATCTTGTTTTA AATGAAGTAATATATTTTATCAAA ACAGGTTTTGTAACCGGGGTGAAGTCGTAACA (SEQ ID NO: 20) Zinderia insecticola CARI spittlebugbacteriocytes ATATAAATAAGAGTTTGATCCTG Clastoptera GCTCAGATTGAACGCTAGCGGTarizonana ATGCTTTACACATGCAAGTCGAA CGACAATATTAAAGCTTGCTTTAATATAAAGTGGCGAACGGGTGA GTAATATATCAAAACGTACCTTA AAGTGGGGGATAACTAATTGAAAAATTAGATAATACCGCATATTAAT CTTAGGATGAAAATAGGAATAAT ATCTTATGCTTTTAGATCGGTTGATATCTGATTAGCTAGTTGGTAG GGTAAATGCTTACCAAGGCAATG ATCAGTAGCTGGTTTTAGCGAATGATCAGCCACACTGGAACTGAG ACACGGTCCAGACTTCTACGGA AGGCAGCAGTGGGGAATATTGGACAATGGGAGAAATCCTGATCCA GCAATACCGCGTGAGTGATGAA GGCCTTAGGGTCGTAAAACTCTTTTGTTAGGAAAGAAATAATTTTAA ATAATATTTAAAATTGATGACGG TACCTAAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGG TAATACGTAGGGTGCAAGCGTTA ATCGGAATTATTGGGCGTAAAGAGTGCGTAGGCTGTTATATAAGAT AGATGTGAAATACTTAAGCTTAA CTTAAGAACTGCATTTATTACTGTTTAACTAGAGTTTATTAGAGAG AAGTGGAATTTTATGTGTAGCAG TGAAATGCGTAGATATATAAAGGAATATCGATGGCGAAGGCAGCT TCTTGGAATAATACTGACGCTGA GGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGT CCACGCCCTAAACTATGTCTACT AGTTATTAAATTAAAAATAAAATTTAGTAACGTAGCTAACGCATTAA GTAGACCGCCTGGGGAGTACGA TCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGC GGTGGATGATGTGGATTAATTCG ATGCAACACGAAAAACCTTACCTACTCTTGACATGTTTGGAATTTT AAAGAAATTTAAAAGTGCTTGAA AAAGAACCAAAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTG TCGTGAGATGTTGGGTTAAGTCC CGCAACGAGCGCAACCCTTGTTATTATTTGCTAATAAAAAGAACTT TAATAAGACTGCCAATGACAAAT TGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGAG TAGGGCTTCACACGTCATACAAT GATATATACAATGGGTAGCAAATTTGTGAAAATGAGCCAATCCTTA AAGTATATCTTAGTTCGGATTGT AGTCTGCAACTCGACTACATGAAGTTGGAATCGCTAGTAATCGCG GATCAGCATGCCGCGGTGAATA CGTTCTCGGGTCTTGTACACACCGCCCGTCACACCATGGAAGTGA TTTTTACCAGAAATTATTTGTTTA ACCTTTATTGGAAAAAAATAATTAAGGTAGAATTCATGACTGGGGT GAAGTCGTAACAAGGTAGCAGT ATCGGAAGGTGCGGCTGGATTACATTTTAAAT (SEQ ID NO: 21) Profftella armatura Diaphorina citri,bacteriomes the Asian citrus psyllid Alpha proteobacteria HodgkiniaCicada bacteriome AATGCTGGCGGCAGGCCTAACA DiceroproctaCATGCAAGTCGAGCGGACAACG semicincta TTCAAACGTTGTTAGCGGCGAACGGGTGAGTAATACGTGAGAATC TACCCATCCCAACGTGATAACAT AGTCAACACCATGTCAATAACGTATGATTCCTGCAACAGGTAAAGA TTTTATCGGGGATGGATGAGCTC ACGCTAGATTAGCTAGTTGGTGAGATAAAAGCCCACCAAGGCCAA GATCTATAGCTGGTCTGGAAGG ATGGACAGCCACATTGGGACTGAGACAAGGCCCAACCCTCTAAG GAGGGCAGCAGTGAGGAATATT GGACAATGGGCGTAAGCCTGATCCAGCCATGCCGCATGAGTGAT TGAAGGTCCAACGGACTGTAAA ACTCTTTTCTCCAGAGATCATAAATGATAGTATCTGGTGATATAAG CTCCGGCCAACTTCGTGCCAGC AGCCGCGGTAATACGAGGGGAGCGAGTATTGTTCGGTTTTATTGG GCGTAAAGGGTGTCCAGGTTGC TAAGTAAGTTAACAACAAAATCTTGAGATTCAACCTCATAACGTTC GGTTAATACTACTAAGCTCGAGC TTGGATAGAGACAAACGGAATTCCGAGTGTAGAGGTGAAATTCGTT GATACTTGGAGGAACACCAGAG GCGAAGGCGGTTTGTCATACCAAGCTGACACTGAAGACACGAAA GCATGGGGAGCAAACAGGATTA GATACCCTGGTAGTCCATGCCCTAAACGTTGAGTGCTAACAGTTC GATCAAGCCACATGCTATGATCC AGGATTGTACAGCTAACGCGTTAAGCACTCCGCCTGGGTATTACG ACCGCAAGGTTAAAACTCAAAG GAATTGACGGAGACCCGCACAAGCGGTGGAGCATGTGGTTTAAT TCGAAGCTACACGAAGAACCTTA CCAGCCCTTGACATACCATGGCCAACCATCCTGGAAACAGGATG TTGTTCAAGTTAAACCCTTGAAA TGCCAGGAACAGGTGCTGCATGGCTGTTGTCAGTTCGTGTCGTGA GATGTATGGTTAAGTCCCAAAAC GAACACAACCCTCACCCATAGTTGCCATAAACACAATTGGGTTCTC TATGGGTACTGCTAACGTAAGTT AGAGGAAGGTGAGGACCACAACAAGTCATCATGGCCCTTATGGG CTGGGCCACACACATGCTACAA TGGTGGTTACAAAGAGCCGCAACGTTGTGAGACCGAGCAAATCT CCAAAGACCATCTCAGTCCGGA TTGTACTCTGCAACCCGAGTACATGAAGTAGGAATCGCTAGTAATC GTGGATCAGCATGCCACGGTGA ATACGTTCTCGGGTCTTGTACACGCCGCCCGTCACACCATGGGAG CTTCGCTCCGATCGAAGTCAAGT TACCCTTGACCACATCTTGGCAAGTGACCGA (SEQ ID NO: 22) Wolbachia sp. wPip Mosquito bacteriomeAAATTTGAGAGTTTGATCCTGGC Culex TCAGAATGAACGCTGGCGGCAG quinquefasciatusGCCTAACACATGCAAGTCGAAC GGAGTTATATTGTAGCTTGCTAT GGTATAACTTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTAC CTAGTAGTACGGAATAATTGTTG GAAACGACAACTAATACCGTATACGCCCTACGGGGGAAAAATTTA TTGCTATTAGATGAGCCTATATT AGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGTAATGATCT ATAGCTGATCTGAGAGGATGATC AGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGC AGCAGTGGGGAATATTGGACAA TGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGC CTTTGGGTTGTAAAGCTCTTTTA GTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAA CTCCGTGCCAGCAGCCGCGGTA ATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGG GCGCGTAGGCTGGTTAATAAGT TAAAAGTGAAATCCCGAGGCTTAACCTTGGAATTGCTTTTAAAACT ATTAATCTAGAGATTGAAAGAGG ATAGAGGAATTCCTGATGTAGAGGTAAAATTCGTAAATATTAGGAG GAACACCAGTGGCGAAGGCGTC TATCTGGTTCAAATCTGACGCTGAAGCGCGAAGGCGTGGGGAGC AAACAGGATTAGATACCCTGGTA GTCCACGCTGTAAACGATGAATGTTAAATATGGGGAGTTTACTTT CTGTATTACAGCTAACGCGTTAA ACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGA ATTGACGGGGACCCGCACAAGC GGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTAC CACTTCTTGACATGAAAATCATA CCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTTACACAAGT GTTGCATGGCTGTCGTCAGCTC GTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT CATCCTTAGTTGCCATCAGGTAA TGCTGAGTACTTTAAGGAAACTGCCAGTGATAAGCTGGAGGAAGG TGGGGATGATGTCAAGTCATCAT GGCCTTTATGGAGTGGGCTACACACGTGCTACAATGGTGTCTACA ATGGGCTGCAAGGTGCGCAAGC CTAAGCTAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCA ACTCGAGTACATGAAGTTGGAAT CGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGG GTCTTGTACACACTGCCCGTCAC GCCATGGGAATTGGTTTCACTCGAAGCTAATGGCCTAACCGCAA GGAAGGAGTTATTTAAAGTGGG ATCAGTGACTGGGGTGAAGTCGTAACAAGGTAGCAGTAGGGGAA TCTGCAGCTGGATTACCTCCTTA (SEQ ID NO: 23)Bacteroidetes Uzinura diaspidicola armoured scale bacteriocytesAAAGGAGATATTCCAACCACACC insects TTCCGGTACGGTTACCTTGTTACGACTTAGCCCTAGTCATCAAGTT TACCTTAGGCAGACCACTGAAG GATTACTGACTTCAGGTACCCCCGACTCCCATGGCTTGACGGGCG GTGTGTACAAGGTTCGAGAACAT ATTCACCGCGCCATTGCTGATGCGCGATTACTAGCGATTCCTGCT TCATAGAGTCGAATTGCAGACTC CAATCCGAACTGAGACTGGTTTTAGAGATTAGCTCCTGATCACCCA GTGGCTGCCCTTTGTAACCAGC CATTGTAGCACGTGTGTAGCCCAAGGCATAGAGGCCATGATGAT TTGACATCATCCCCACCTTCCTC ACAGTTTACACCGGCAGTTTTGTTAGAGTCCCCGGCTTTACCCGA TGGCAACTAACAATAGGGGTTG CGCTCGTTATAGGACTTAACCAAACACTTCACAGCACGAACTGAA GACAACCATGCAGCACCTTGTAA TACGTCGTATAGACTAAGCTGTTTCCAGCTTATTCGTAATACATTTA AGCCTTGGTAAGGTTCCTCGCG TATCATCGAATTAAACCACATGCTCCACCGCTTGTGCGAACCCCC GTCAATTCCTTTGAGTTTCAATC TTGCGACTGTACTTCCCAGGTGGATCACTTATCGCTTTCGCTAAG CCACTGAATATCGTTTTTCCAAT AGCTAGTGATCATCGTTTAGGGCGTGGACTACCAGGGTATCTAATC CTGTTTGCTCCCCACGCTTTCGT GCACTGAGCGTCAGTAAAGATTTAGCAACCTGCCTTCGCTATCGG TGTTCTGTATGATATCTATGCATT TCACCGCTACACCATACATTCCAGATGCTCCAATCTTACTCAAGTT TACCAGTATCAATAGCAATTTTA CAGTTAAGCTGTAAGCTTTCACTACTGACTTAATAAACAGCCTACA CACCCTTTAAACCCAATAAATCC GAATAACGCTTGTGTCATCCGTATTGCCGCGGCTGCTGGCACGGA ATTAGCCGACACTTATTCGTATA GTACCTTCAATCTCCTATCACGTAAGATATTTTATTTCTATACAAAA GCAGTTTACAACCTAAAAGACCT TCATCCTGCACGCGACGTAGCTGGTTCAGAGTTTCCTCCATTGAC CAATATTCCTCACTGCTGCCTCC CGTAGGAGTCTGGTCCGTGTCTCAGTACCAGTGTGGAGGTACAC CCTCTTAGGCCCCCTACTGATCA TAGTCTTGGTAGAGCCATTACCTCACCAACTAACTAATCAAACGCA GGCTCATCTTTTGCCACCTAAGT TTTAATAAAGGCTCCATGCAGAAACTTTATATTATGGGGGATTAAT CAGAATTTCTTCTGGCTATACCC CAGCAAAAGGTAGATTGCATACGTGTTACTCACCCATTCGCCGGT CGCCGACAAATTAAAAATTTTTC GATGCCCCTCGACTTGCATGTGTTAAGCTCGCCGCTAGCGTTAAT TCTGAGCCAGGATCAAACTCTTC GTTGTAG (SEQ ID NO: 24)Sulcia muelleri Blue-Green bacteriocytes CTCAGGATAAACGCTAGCGGAGSharpshooter GGCTTAACACATGCAAGTCGAG and several otherGGGCAGCAAAAATAATTATTTTT leafhopper GGCGACCGGCAAACGGGTGAGT speciesAATACATACGTAACTTTCCTTAT GCTGAGGAATAGCCTGAGGAAA CTTGGATTAATACCTCATAATACAATTTTTTAGAAAGAAAAATTGTT AAAGTTTTATTATGGCATAAGAT AGGCGTATGTCCAATTAGTTAGTTGGTAAGGTAATGGCTTACCAAG ACGATGATTGGTAGGGGGCCTG AGAGGGGCGTTCCCCCACATTGGTACTGAGACACGGACCAAACT TCTACGGAAGGCTGCAGTGAGG AATATTGGTCAATGGAGGAAACTCTGAACCAGCCACTCCGCGTGC AGGATGAAAGAAAGCCTTATTGG TTGTAAACTGCTTTTGTATATGAATAAAAAATTCTAATTATAGAAATA ATTGAAGGTAATATACGAATAAG TATCGACTAACTCTGTGCCAGCAGTCGCGGTAAGACAGAGGATAC AAGCGTTATCCGGATTTATTGGG TTTAAAGGGTGCGTAGGCGGTTTTTAAAGTCAGTAGTGAAATCTT AAAGCTTAACTTTAAAAGTGCTA TTGATACTGAAAAACTAGAGTAAGGTTGGAGTAACTGGAATGTGT GGTGTAGCGGTGAAATGCATAG ATATCACACAGAACACCGATAGCGAAAGCAAGTTACTAACCCTATA CTGACGCTGAGTCACGAAAGCA TGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAA ACGATGATCACTAACTATTGGGT TTTATACGTTGTAATTCAGTGGTGAAGCGAAAGTGTTAAGTGATC CACCTGAGGAGTACGACCGCAA GGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAATCGGTGG AGCATGTGGTTTAATTCGATGAT ACACGAGGAACCTTACCAAGACTTAAATGTACTACGAATAAATTG GAAACAATTTAGTCAAGCGACG GAGTACAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGG TGTAAGGTTAAGTCCTTTAAACG AGCGCAACCCTTATTATTAGTTGCCATCGAGTAATGTCAGGGGAC TCTAATAAGACTGCCGGCGCAA GCCGAGAGGAAGGTGGGGATGACGTCAAATCATCACGGCCCTTA CGTCTTGGGCCACACACGTGCT ACAATGATCGGTACAAAAGGGAGCGACTGGGTGACCAGGAGCAA ATCCAGAAAGCCGATCTAAGTTC GGATTGGAGTCTGAAACTCGACTCCATGAAGCTGGAATCGCTAGT AATCGTGCATCAGCCATGGCAC GGTGAATATGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCA TGGAAGTTGGAAGTACCTAAAGT TGGTTCGCTACCTAAGGTAAGTCTAATAACTGGGGCTAAGTCGTAA CAAGGTA (SEQ ID NO: 25) Yeast likeSymbiotaphrina buchneri Anobiid beetles mycetome AGATTAAGCCATGCAAGTCTAAGvoucher JCM9740 Stegobium between the TATAAGNAATCTATACNGTGAAA paniceumforegut and CTGCGAATGGCTCATTAAATCAG midgut TTATCGTTTATTTGATAGTACCTTACTACATGGATAACCGTGGTAAT TCTAGAGCTAATACATGCTAAAA ACCCCGACTTCGGAAGGGGTGTATTTATTAGATAAAAAACCAATG CCCTTCGGGGCTCCTTGGTGAT TCATGATAACTTAACGAATCGCATGGCCTTGCGCCGGCGATGGTT CATTCAAATTTCTGCCCTATCAA CTTTCGATGGTAGGATAGTGGCCTACCATGGTTTTAACGGGTAAC GGGGAATTAGGGTTCGATTCCG GAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGC AGGCGCGCAAATTACCCAATCC CGACACGGGGAGGTAGTGACAATAAATACTGATACAGGGCTCTTT TGGGTCTTGTAATTGGAATGAGT ACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGG TGCCAGCAGCCGCGGTAATTCC AGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTA GTTGAACCTTGGGCCTGGCTGG CCGGTCCGCCTAACCGCGTGTACTGGTCCGGCCGGGCCTTTCCT TCTGGGGAGCCGCATGCCCTTC ACTGGGTGTGTCGGGGAACCAGGACTTTTACTTTGAAAAAATTAGA GTGTTCAAAGCAGGCCTATGCT CGAATACATTAGCATGGAATAATAGAATAGGACGTGCGGTTCTATT TTGTTGGTTTCTAGGACCGCCGT AATGATTAATAGGGATAGTCGGGGGCATCAGTATTCAATTGTCAGA GGTGAAATTCTTGGATTTATTGA AGACTAACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATC AGTGAACGAAAGTTAGGGGATC GAAGACGATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCG ACTAGGGATCGGGCGATGTTAT TATTTTGACTCGCTCGGCACCTTACGAGAAATCAAAGTCTTTGGGT TCTGGGGGGAGTATGGTCGCAA GGCTGAAACTTAAAGAAATTGACGGAAGGGCACCACCAGGAGTG GAGCCTGCGGCTTAATTTGACTC AACACGGGGAAACTCACCAGGTCCAGACACATTAAGGATTGACAG ATTGAGAGCTCTTTCTTGATTAT GTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGT CTGCTTAATTGCGATAACGAACG AGACCTTAACCTGCTAAATAGCCCGGTCCGCTTTGGCGGGCCGCT GGCTTCTTAGAGGGACTATCGG CTCAAGCCGATGGAAGTTTGAGGCAATAACAGGTCTGTGATGCC CTTAGATGTTCTGGGCCGCACG CGCGCTACACTGACAGAGCCAACGAGTAAATCACCTTGGCCGGA AGGTCTGGGTAATCTTGTTAAAC TCTGTCGTGCTGGGGATAGAGCATTGCAATTATTGCTCTTCAACG AGGAATTCCTAGTAAGCGCAAGT CATCAGCTTGCGCTGATTACGTCCCTGCCCTTTGTACACACCGCC CGTCGCTACTACCGATTGAATG GCTCAGTGAGGCCTTCGGACTGGCACAGGGACGTTGGCAACGAC GACCCAGTGCCGGAAAGTTGGT CAAACTTGGTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCG TAGGTGAACCTGCGGAAGGATC ATTA (SEQ ID NO: 26)Symbiotaphrina kochii Anobiid beetles mycetome TACCTGGTTGATTCTGCCAGTAGvoucher CBS 589.63 Lasioderma TCATATGCTTGTCTCAAAGATTA serricomeAGCCATGCAAGTCTAAGTATAAG CAATCTATACGGTGAAACTGCGA ATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTACCTTACTACA TGGATAACCGTGGTAATTCTAGA GCTAATACATGCTAAAAACCTCGACTTCGGAAGGGGTGTATTTATT AGATAAAAAACCAATGCCCTTCG GGGCTCCTTGGTGATTCATGATAACTTAACGAATCGCATGGCCTTG CGCCGGCGATGGTTCATTCAAA TTTCTGCCCTATCAACTTTCGATGGTAGGATAGTGGCCTACCATG GTTTCAACGGGTAACGGGGAAT TAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACA TCCAAGGAAGGCAGCAGGCGCG CAAATTACCCAATCCCGACACGGGGAGGTAGTGACAATAAATACT GATACAGGGCTCTTTTGGGTCTT GTAATTGGAATGAGTACAATTTAAATCCCTTAACGAGGAACAATTG GAGGGCAAGTCTGGTGCCAGCA GCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCA GTTAAAAAGCTCGTAGTTGAACC TTGGGCCTGGCTGGCCGGTCCGCCTAACCGCGTGTACTGGTCCG GCCGGGCCTTTCCTTCTGGGGA GCCGCATGCCCTTCACTGGGTGTGTCGGGGAACCAGGACTTTTA CTTTGAAAAAATTAGAGTGTTCA AAGCAGGCCTATGCTCGAATACATTAGCATGGAATAATAGAATAG GACGTGTGGTTCTATTTTGTTGG TTTCTAGGACCGCCGTAATGATTAATAGGGATAGTCGGGGGCATC AGTATTCAATTGTCAGAGGTGAA ATTCTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAA GGATGTTTTCATTAATCAGTGAA CGAAAGTTAGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTA ACCATAAACTATGCCGACTAGG GATCGGGCGATGTTATTATTTTGACTCGCTCGGCACCTTACGAGA AATCAAAGTCTTTGGGTTCTGGG GGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAATTGACGGAAGG GCACCACCAGGAGTGGAGCCTG CGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACA CATTAAGGATTGACAGATTGAGA GCTCTTTCTTGATTATGTGGGTGGTGGTGCATGGCCGTTCTTAGTT GGTGGAGTGATTTGTCTGCTTAA TTGCGATAACGAACGAGACCTTAACCTGCTAAATAGCCCGGTCCG CTTTGGCGGGCCGCTGGCTTCT TAGAGGGACTATCGGCTCAAGCCGATGGAAGTTTGAGGCAATAA CAGGTCTGTGATGCCCTTAGAT GTTCTGGGCCGCACGCGCGCTACACTGACAGAGCCAACGAGTAC ATCACCTTGGCCGGAAGGTCTG GGTAATCTTGTTAAACTCTGTCGTGCTGGGGATAGAGCATTGCAA TTATTGCTCTTCAACGAGGAATT CCTAGTAAGCGCAAGTCATCAGCTTGCGCTGATTACGTCCCTGC CCTTTGTACACACCGCCCGTCG CTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGCACAG GGACGTTGGCAACGACGACCCA GTGCCGGAAAGTTCGTCAAACTTGGTCATTTAGAGGAAGNNNAAG TCGTAACAAGGTTTCCGTAGGTG AACCTGCGGAAGGATCATTA(SEQ ID NO: 27) Primary extracelullar symbiont Host location 16 rRNAfenitrothion-degrading bacteria Burkholderia sp. SFA1 Riptortus GutAGTTTGATCCTGGCTCAGATTGA pedestris ACGCTGGCGGCATGCCTTACACATGCAAGTCGAACGGCAGCACG GGGGCAACCCTGGTGGCGAGT GGCGAACGGGTGAGTAATACATCGGAACGTGTCCTGTAGTGGGG GATAGCCCGGCGAAAGCCGGAT TAATACCGCATACGACCTAAGGGAGAAAGCGGGGGATCTTCGGA CCTCGCGCTATAGGGGCGGCCG ATGGCAGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGA CGATCTGTAGCTGGTCTGAGAG GACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTAC GGGAGGCAGCAGTGGGGAATTT TGGACAATGGGGGCAACCCTGATCCAGCAATGCCGCGTGTGTGA AGAAGGCTTCGGGTTGTAAAGC ACTTTTGTCCGGAAAGAAAACTTCGTCCCTAATATGGATGGAGGA TGACGGTACCGGAAGAATAAGC ACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGC GAGCGTTAATCGGAATTACTGG GCGTAAAGCGTGCGCAGGCGGTCTGTTAAGACCGATGTGAAATCC CCGGGCTTAACCTGGGAACTGC ATTGGTGACTGGCAGGCTTTGAGTGTGGCAGAGGGGGGTAGAAT TCCACGTGTAGCAGTGAAATGC GTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGG CCAACTACTGACGCTCATGCAC GAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACG CCCTAAACGATGTCAACTAGTTG TTGGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGA CCGCCTGGGGAGTACGGTCGCA AGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTG GATGATGTGGATTAATTCGATGC AACGCGAAAAACCTTACCTACCCTTGACATGGTCGGAACCCTGCT GAAAGGTGGGGGTGCTCGAAAG AGAACCGGCGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTC GTGAGATGTTGGGTTAAGTCCC GCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCACTC TAAGGAGACTGCCGGTGACAAA CCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATG GGTAGGGCTTCACACGTCATAC AATGGTCGGAACAGAGGGTTGCCAAGCCGCGAGGTGGAGCCAAT CCCAGAAAACCGATCGTAGTCC GGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAG TAATCGCGGATCAGCATGCCGC GGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCA TGGGAGTGGGTTTCACCAGAAG TAGGTAGCCTAACCGCAAGGAGGGCGCTTACCACGGTGGGATTC ATGACTGGGGTGAAGTCGTAAC AAGGTAGC (SEQ ID NO: 28)Burkholderia sp. KM-A Riptortus Gut GCAACCCTGGTGGCGAGTGGCG pedestrisAACGGGTGAGTAATACATCGGA ACGTGTCCTGTAGTGGGGGATA GCCCGGCGAAAGCCGGATTAATACCGCATACGATCTACGGAAGA AAGCGGGGGATCCTTCGGGACC TCGCGCTATAGGGGCGGCCGATGGCAGATTAGCTAGTTGGTGGG GTAAAGGCCTACCAAGGCGACG ATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGA GACACGGCCCAGACTCCTACGG GAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGATC CAGCAATGCCGCGTGTGTGAAG AAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAAACGTC TTGGTTAATACCTGAGGCGGAT GACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCAG CCGCGGTAATACGTAGGGTGCG AGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTC TGTTAAGACCGATGTGAAATCCC CGGGCTTAACCTGGGAACTGCATTGGTGACTGGCAGGCTTTGAG TGTGGCAGAGGGGGGTAGAATT CCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGA TGGCGAAGGCAGCCCCCTGGG CCAACACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGA TTAGATACCCTGGTAGTCCACGC CCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAAC GTAGCTAACGCGTGAAGTTGAC CGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGAC GGGGACCCGCACAAGCGGTGG ATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCT TGACATGGTCGGAAGTCTGCTG AGAGGTGGACGTGCTCGAAAGAGAACCGGCGCACAGGTGCTGCA TGGCTGTCGTCAGCTCGTGTCG TGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCT TAGTTGCTACGCAAGAGCACTCT AAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGT CAAGTCCTCATGGCCCTTATGG GTAGGGCTTCACACGTCATACAATGGTCGGAACAGAGGGTTGCCA AGCCGCGAGGTGGAGCCAATCC CAGAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTG CGTGAAGCTGGAATCGCTAGTA ATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTA CACACCGCCCGTCACACCATGG GAGTGGGTTTCACCAGAAGTAGGTAGCCTAACCGCAAGGAGGGC GCTTACCACGGTGGGATTCATG ACTGGGGTGAAGT(SEQ ID NO: 29) Burkholderia sp. KM-G Riptortus GutGCAACCCTGGTGGCGAGTGGCG pedestris AACGGGTGAGTAATACATCGGAACGTGTCCTGTAGTGGGGGATA GCCCGGCGAAAGCCGGATTAAT ACCGCATACGACCTAAGGGAGAAAGCGGGGGATCTTCGGACCTC GCGCTATAGGGGCGGCCGATG GCAGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGA TCTGTAGCTGGTCTGAGAGGAC GACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGG AGGCAGCAGTGGGGAATTTTGG ACAATGGGGGCAACCCTGATCCAGCAATGCCGCGTGTGTGAAGA AGGCCTTCGGGTTGTAAAGCAC TTTTGTCCGGAAAGAAAACTTCGAGGTTAATACCCTTGGAGGATGA CGGTACCGGAAGAATAAGCACC GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAG CGTTAATCGGAATTACTGGGCGT AAAGCGTGCGCAGGCGGTCTGTTAAGACCGATGTGAAATCCCCG GGCTTAACCTGGGAACTGCATT GGTGACTGGCAGGCTTTGAGTGTGGCAGAGGGGGGTAGAATTCC ACGTGTAGCAGTGAAATGCGTA GAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCC AACACTGACGCTCATGCACGAA AGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCC CTAAACGATGTCAACTAGTTGTT GGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACC GCCTGGGGAGTACGGTCGCAAG ATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAT GATGTGGATTAATTCGATGCAAC GCGAAAAACCTTACCTACCCTTGACATGGTCGGAAGTCTGCTGAG AGGTGGACGTGCTCGAAAGAGA ACCGGCGCACAGGTGCTGCATGGCTGTC GTCAGCTCGTGTCGTGAGATGT TGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTA CGCAAGAGCACTCTAAGGAGAC TGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCT CATGGCCCTTATGGGTAGGGCT TCACACGTCATACAATGGTCGGAACAGAGGGTTGCCAAGCCGCGA GGTGGAGCCAATCCCAGAAAAC CGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGC TGGAATCGCTAGTAATCGCGGA TCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCG CCCGTCACACCATGGGAGTGGG TTTCACCAGAAGTAGGTAGCCTAACCTGCAAAGGAGGGCGCTTAC CACG (SEQ ID NO: 30) Bees Snodgrassella alviHoneybee (Apis Ileum GAGAGTTTGATCCTGGCTCAGAT mellifera) andTGAACGCTGGCGGCATGCCTTA Bombus spp. CACATGCAAGTCGAACGGCAGCACGGAGAGCTTGCTCTCTGGTG GCGAGTGGCGAACGGGTGAGTA ATGCATCGGAACGTACCGAGTAATGGGGGATAACTGTCCGAAAG GATGGCTAATACCGCATACGCC CTGAGGGGGAAAGCGGGGGATCGAAAGACCTCGCGTTATTTGAG CGGCCGATGTTGGATTAGCTAG TTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCGGGTC TGAGAGGATGATCCGCCACATT GGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGG GAATTTTGGACAATGGGGGGAA CCCTGATCCAGCCATGCCGCGTGTCTGAAGAAGGCCTTCGGGTT GTAAAGGACTTTTGTTAGGGAAG AAAAGCCGGGTGTTAATACCATCTGGTGCTGACGGTACCTAAAGA ATAAGCACCGGCTAACTACGTG CCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAAT TACTGGGCGTAAAGCGAGCGCA GACGGTTAATTAAGTCAGATGTGAAATCCCCGAGCTCAACTTGGG ACGTGCATTTGAAACTGGTTAAC TAGAGTGTGTCAGAGGGAGGTAGAATTCCACGTGTAGCAGTGAAA TGCGTAGAGATGTGGAGGAATA CCGATGGCGAAGGCAGCCTCCTGGGATAACACTGACGTTCATGCT CGAAAGCGTGGGTAGCAAACAG GATTAGATACCCTGGTAGTCCACGCCCTAAACGATGACAATTAGCT GTTGGGACACTAGATGTCTTAGT AGCGAAGCTAACGCGTGAAATTGTCCGCCTGGGGAGTACGGTCG CAAGATTAAAACTCAAAGGAATT GACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGAT GCAACGCGAAGAACCTTACCTG GTCTTGACATGTACGGAATCTCTTAGAGATAGGAGAGTGCCTTCG GGAACCGTAACACAGGTGCTGC ATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCC GCAACGAGCGCAACCCTTGTCA TTAGTTGCCATCATTAAGTTGGGCACTCTAATGAGACTGCCGGTG ACAAACCGGAGGAAGGTGGGGA TGACGTCAAGTCCTCATGGCCCTTATGACCAGGGCTTCACACGTC ATACAATGGTCGGTACAGAGGG TAGCGAAGCCGCGAGGTGAAGCCAATCTCAGAAAGCCGATCGTA GTCCGGATTGCACTCTGCAACT CGAGTGCATGAAGTCGGAATCGCTAGTAATCGCAGGTCAGCATAC TGCGGTGAATACGTTCCCGGGT CTTGTACACACCGCCCGTCACACCATGGGAGTGGGGGATACCAG AATTGGGTAGACTAACCGCAAG GAGGTCGCTTAACACGGTATGCTTCATGACTGGGGTGAAGTCGT AACAAGGTAGCCGTAG (SEQ ID NO: 31)Gilliamella apicola honeybee (Apis Ileum TTAAATTGAAGAGTTTGATCATGmellifera) and GCTCAGATTGAACGCTGGCGGC Bombus spp.AGGCTTAACACATGCAAGTCGAA CGGTAACATGAGTGCTTGCACTT GATGACGAGTGGCGGACGGGTGAGTAAAGTATGGGGATCTGCC GAATGGAGGGGGACAACAGTTG GAAACGACTGCTAATACCGCATAAAGTTGAGAGACCAAAGCATGG GACCTTCGGGCCATGCGCCATT TGATGAACCCATATGGGATTAGCTAGTTGGTAGGGTAATGGCTTAC CAAGGCGACGATCTCTAGCTGG TCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAG ACTCCTACGGGAGGCAGCAGTG GGGAATATTGCACAATGGGGGAAACCCTGATGCAGCCATGCCGC GTGTATGAAGAAGGCCTTCGGG TTGTAAAGTACTTTCGGTGATGAGGAAGGTGGTGTATCTAATAGG TGCATCAATTGACGTTAATTACA GAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACG GAGGGTGCGAGCGTTAATCGGA ATGACTGGGCGTAAAGGGCATGTAGGCGGATAATTAAGTTAGGTG TGAAAGCCCTGGGCTCAACCTA GGAATTGCACTTAAAACTGGTTAACTAGAGTATTGTAGAGGAAGGT AGAATTCCACGTGTAGCGGTGA AATGCGTAGAGATGTGGAGGAATACCGGTGGCGAAGGCGGCCTT CTGGACAGATACTGACGCTGAG ATGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTC CACGCTGTAAACGATGTCGATTT GGAGTTTGTTGCCTAGAGTGATGGGCTCCGAAGCTAACGCGATA AATCGACCGCCTGGGGAGTACG GCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAG CGGTGGAGCATGTGGTTTAATTC GATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACAGAATC TTGCAGAGATGCGGGAGTGCCT TCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGT GTTGTGAAATGTTGGGTTAAGTC CCGCAACGAGCGCAACCCTTATCCTTTGTTGCCATCGGTTAGGCC GGGAACTCAAAGGAGACTGCCG TTGATAAAGCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGG CCCTTACGACCAGGGCTACACA CGTGCTACAATGGCGTATACAAAGGGAGGCGACCTCGCGAGAGC AAGCGGACCTCATAAAGTACGT CTAAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGA ATCGCTAGTAATCGTGAATCAGA ATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGT CACACCATGGGAGTGGGTTGCA CCAGAAGTAGATAGCTTAACCTTCGGGAGGGCGTTTACCACGGTG TGGTCCATGACTGGGGTGAAGT CGTAACAAGGTAACCGTAGGGGAACCTGCGGTTGGATCACCTCC TTAC (SEQ ID NO: 32) Bartonella apishoneybee (Apis Gut AAGCCAAAATCAAATTTTCAACT mellifera)TGAGAGTTTGATCCTGGCTCAGA ACGAACGCTGGCGGCAGGCTTA ACACATGCAAGTCGAACGCACTTTTCGGAGTGAGTGGCAGACGGG TGAGTAACGCGTGGGAATCTAC CTATTTCTACGGAATAACGCAGAGAAATTTGTGCTAATACCGTATA CGTCCTTCGGGAGAAAGATTTAT CGGAGATAGATGAGCCCGCGTTGGATTAGCTAGTTGGTGAGGTA ATGGCCCACCAAGGCGACGATC CATAGCTGGTCTGAGAGGATGACCAGCCACATTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAG GCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAG CCATGCCGCGTGAGTGATGAAG GCCCTAGGGTTGTAAAGCTCTTTCACCGGTGAAGATAATGACGGT AACCGGAGAAGAAGCCCCGGCT AACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTT GTTCGGATTTACTGGGCGTAAA GCGCACGTAGGCGGATATTTAAGTCAGGGGTGAAATCCCGGGGC TCAACCCCGGAACTGCCTTTGAT ACTGGATATCTTGAGTATGGAAGAGGTAAGTGGAATTCCGAGTGT AGAGGTGAAATTCGTAGATATTC GGAGGAACACCAGTGGCGAAGGCGGCTTACTGGTCCATTACTGAC GCTGAGGTGCGAAAGCGTGGG GAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGAT GAATGTTAGCCGTTGGACAGTTT ACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGG AGTACGGTCGCAAGATTAAAACT CAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT TTAATTCGAAGCAACGCGCAGAA CCTTACCAGCCCTTGACATCCCGATCGCGGATGGTGGAGACACC GTCTTTCAGTTCGGCTGGATCG GTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATG TTGGGTTAAGTCCCGCAACGAG CGCAACCCTCGCCCTTAGTTGCCATCATTTAGTTGGGCACTCTAA GGGGACTGCCGGTGATAAGCCG AGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGG CTGGGCTACACACGTGCTACAA TGGTGGTGACAGTGGGCAGCGAGACCGCGAGGTCGAGCTAATCT CCAAAAGCCATCTCAGTTCGGAT TGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCG TGGATCAGCATGCCACGGTGAA TACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAG TTGGTTTTACCCGAAGGTGCTGT GCTAACCGCAAGGAGGCAGGCAACCACGGTAGGGTCAGCGACTG GGGTGAAGTCGTAACAAGGTAG CCGTAGGGGAACCTGCGGCTGGATCACCTCCTTTCTAAGGAAGAT GAAGAATTGGAA (SEQ ID NO: 33) Parasaccharibacterhoneybee (Apis Gut CTACCATGCAAGTCGCACGAAA apium mellifera)CCTTTCGGGGTTAGTGGCGGAC GGGTGAGTAACGCGTTAGGAAC CTATCTGGAGGTGGGGGATAACATCGGGAAACTGGTGCTAATAC CGCATGATGCCTGAGGGCCAAA GGAGAGATCCGCCATTGGAGGGGCCTGCGTTCGATTAGCTAGTTG GTTGGGTAAAGGCTGACCAAGG CGATGATCGATAGCTGGTTTGAGAGGATGATCAGCCACACTGGG ACTGAGACACGGCCCAGACTCC TACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCC TGATCCAGCAATGCCGCGTGTG TGAAGAAGGTCTTCGGATTGTAAAGCACTTTCACTAGGGAAGATGA TGACGGTACCTAGAGAAGAAGC CCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGC TAGCGTTGCTCGGAATGACTGG GCGTAAAGGGCGCGTAGGCTGTTTGTACAGTCAGATGTGAAATCC CCGGGCTTAACCTGGGAACTGC ATTTGATACGTGCAGACTAGAGTCCGAGAGAGGGTTGTGGAATTC CCAGTGTAGAGGTGAAATTCGTA GATATTGGGAAGAACACCGGTTGCGAAGGCGGCAACCTGGCTNN NNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNGAGCTAACGCGTTAAGCACA CCGCCTGGGGAGTACGGCCGC AAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT GGAGCATGTGGTTTAATTCGAAG CAACGCGCAGAACCTTACCAGGGCTTGCATGGGGAGGCTGTATT CAGAGATGGATATTTCTTCGGAC CTCCCGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGA GATGTTGGGTTAAGTCCCGCAA CGAGCGCAACCCTTGTCTTTAGTTGCCATCACGTCTGGGTGGGCA CTCTAGAGAGACTGCCGGTGAC AAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCT TATGTCCTGGGCTACACACGTG CTACAATGGCGGTGACAGAGGGATGCTACATGGTGACATGGTGCT GATCTCAAAAAACCGTCTCAGTT CGGATTGTACTCTGCAACTCGAGTGCATGAAGGTGGAATCGCTA GTAATCGCGGATCAGCATGCCG CGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC ATGGGAGTTGGTTTGACCTTAAG CCGGTGAGCGAACCGCAAGGAACGCAGCCGACCACCGGTTCGGG TTCAGCGACTGGGGGA (SEQ ID NO: 34)Lactobacillus kunkeei honeybee (Apis Gut TTCCTTAGAAAGGAGGTGATCCAmellifera) GCCGCAGGTTCTCCTACGGCTA CCTTGTTACGACTTCACCCTAATCATCTGTCCCACCTTAGACGACT AGCTCCTAAAAGGTTACCCCATC GTCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTA CAAGGCCCGGGAACGTATTCAC CGTGGCATGCTGATCCACGATTACTAGTGATTCCAACTTCATGCA GGCGAGTTGCAGCCTGCAATCC GAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGC GACTCGTTGTACCATCCATTGTA GCACGTGTGTAGCCCAGCTCATAAGGGGCATGATGATTTGACGT CGTCCCCACCTTCCTCCGGTTTA TCACCGGCAGTCTCACTAGAGTGCCCAACTAAATGCTGGCAACTA ATAATAAGGGTTGCGCTCGTTGC GGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCAT GCACCACCTGTCATTCTGTCCCC GAAGGGAACGCCCAATCTCTTGGGTTGGCAGAAGATGTCAAGAG CTGGTAAGGTTCTTCGCGTAGC ATCGAATTAAACCACATGCTCCACCACTTGTGCGGGCCCCCGTCA ATTCCTTTGAGTTTCAACCTTGC GGTCGTACTCCCCAGGCGGAATACTTAATGCGTTAGCTGCGGCA CTGAAGGGCGGAAACCCTCCAA CACCTAGTATTCATCGTTTACGGCATGGACTACCAGGGTATCTAAT CCTGTTCGCTACCCATGCTTTCG AGCCTCAGCGTCAGTAACAGACCAGAAAGCCGCCTTCGCCACTG GTGTTCTTCCATATATCTACGCA TTTCACCGCTACACATGGAGTTCCACTTTCCTCTTCTGTACTCAAG TTTTGTAGTTTCCACTGCACTTC CTCAGTTGAGCTGAGGGCTTTCACAGCAGACTTACAAAACCGCCT GCGCTCGCTTTACGCCCAATAAA TCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCA CGTAGTTAGCCGTGGCTTTCTG GTTAAATACCGTCAAAGTGTTAACAGTTACTCTAACACTTGTTCTT CTTTAACAACAGAGTTTTACGAT CCGAAAACCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTC GTCCATTGTGGAAGATTCCCTAC TGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAATGT GGCCGATTACCCTCTCAGGTCG GCTACGTATCATCGTCTTGGTGGGCTTTTATCTCACCAACTAACTA ATACGGCGCGGGTCCATCCCAA AGTGATAGCAAAGCCATCTTTCAAGTTGGAACCATGCGGTTCCAA CTAATTATGCGGTATTAGCACTT GTTTCCAAATGTTATCCCCCGCTTCGGGGCAGGTTACCCACGTGT TACTCACCAGTTCGCCACTCGCT CCGAATCCAAAAATCATTTATGCAAGCATAAAATCAATTTGGGAGA ACTCGTTCGACTTGCATGTATTA GGCACGCCGCCAGCGTTCGTCCTGAGCCAGGATCAAACTCTCATC TTAA (SEQ ID NO: 35) Lactobacillus Firm-4honeybee (Apis Gut ACGAACGCTGGCGGCGTGCCTA mellifera)ATACATGCAAGTCGAGCGCGGG AAGTCAGGGAAGCCTTCGGGTG GAACTGGTGGAACGAGCGGCGGATGGGTGAGTAACACGTAGGT AACCTGCCCTAAAGCGGGGGAT ACCATCTGGAAACAGGTGCTAATACCGCATAAACCCAGCAGTCAC ATGAGTGCTGGTTGAAAGACGG CTTCGGCTGTCACTTTAGGATGGACCTGCGGCGTATTAGCTAGTT GGTGGAGTAACGGTTCACCAAG GCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGG GACTGAGACACGGCCCAAACTC CTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTC TGATGGAGCAACGCCGCGTGGA TGAAGAAGGTCTTCGGATCGTAAAATCCTGTTGTTGAAGAAGAACG GTTGTGAGAGTAACTGCTCATAA CGTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAG CAGCCGCGGTAATACGTAGGTG GCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGAGCGCAGGCG GTCTTTTAAGTCTGAATGTGAAA GCCCTCAGCTTAACTGAGGAAGAGCATCGGAAACTGAGAGACTT GAGTGCAGAAGAGGAGAGTGGA ACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACAC CAGTGGCGAAGGCGGCTCTCTG GTCTGTTACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAG GATTAGATACCCTGGTAGTCCAT GCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTC AGTGCTGCAGCTAACGCATTAA GCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGG AATTGACGGGGGCCCGCACAAG CGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTAC CAGGTCTTGACATCTCCTGCAAG CCTAAGAGATTAGGGGTTCCCTTCGGGGACAGGAAGACAGGTGGT GCATGGTTGTCGTCAGCTCGTG TCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTT ACTAGTTGCCAGCATTAAGTTGG GCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGG GACGACGTCAAATCATCATGCC CCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAATG AGAAGCGAACTCGCGAGGGGAA GCTGATCTCTGAAAACCATTCTCAGTTCGGATTGCAGGCTGCAAC TCGCCTGCATGAAGCTGGAATC GCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGG GCCTTGTACACACCGCCC (SEQ ID NO: 36) Silk wormEnterococcus Bombyx mor Gut AGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACT TCACCCCAATCATCTATCCCACC TTAGGCGGCTGGCTCCAAAAAGGTTACCTCACCGACTTCGGGTG TTACAAACTCTCGTGGTGTGACG GGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGC TGATCCGCGATTACTAGCGATTC CGGCTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAG AAGCTTTAAGAGATTTGCATGAC CTCGCGGTCTAGCGACTCGTTGTACTTCCCATTGTAGCACGTGTG TAGCCCAGGTCATAAGGGGCAT GATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCA GTCTCGCTAGAGTGCCCAACTA AATGATGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTA ACCCAACATCTCACGACACGAG CTGACGACAACCATGCACCACCTGTCACTTTGTCCCCGAAGGGA AAGCTCTATCTCTAGAGTGGTCA AAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTA AACCACATGCTCCACCGCTTGT GCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTAC TCCCCAGGCGGAGTGCTTAATG CGTTTGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGC ACTCATCGTTTACGGCGTGGACT ACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCA GCGTCAGTTACAGACCAGAGAG CCGCCTTCGCCACTGGTGTTCCTCCATATATCTACGCATTTCACC GCTACACATGGAATTCCACTCTC CTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGACCCTCCCCGGTT GAGCCGGGGGCTTTCACATCAG ACTTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGA CAACGCTTGCCACCTACGTATTA CCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGATA CCGTCAGGGGACGTTCAGTTAC TAACGTCCTTGTTCTTCTCTAACAACAGAGTTTTACGATCCGAAAA CCTTCTTCACTCACGCGGCGTT GCTCGGTCAGACTTTCGTCCATTGCCGAAGATTCCCTACTGCTGC CTCCCGTAGGAGTCTGGGCCGT GTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATG CATCGTGGCCTTGGTGAGCCGT TACCTCACCAACTAGCTAATGCACCGCGGGTCCATCCATCAGCGA CACCCGAAAGCGCCTTTCACTCT TATGCCATGCGGCATAAACTGTTATGCGGTATTAGCACCTGTTTCC AAGTGTTATCCCCCTCTGATGGG TAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCCTCTTTCCAA TTGAGTGCAAGCACTCGGGAGG AAAGAAGCGTTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTC GTCCTGAGCCAGGATCAAACTC T (SEQ ID NO: 37) DelftiaBombyx mori Gut CAGAAAGGAGGTGATCCAGCCG CACCTTCCGATACGGCTACCTTGTTACGACTTCACCCCAGTCACGA ACCCCGCCGTGGTAAGCGCCCT CCTTGCGGTTAGGCTACCTACTTCTGGCGAGACCCGCTCCCATGG TGTGACGGGCGGTGTGTACAAG ACCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTA GCGATTCCGACTTCACGCAGTC GAGTTGCAGACTGCGATCCGGACTACGACTGGTTTTATGGGATTA GCTCCCCCTCGCGGGTTGGCAA CCCTCTGTACCAGCCATTGTATGACGTGTGTAGCCCCACCTATAA GGGCCATGAGGACTTGACGTCA TCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGC TCAACTGAATGTAGCAACTAATG ACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGAC ACGAGCTGACGACAGCCATGCA GCACCTGTGTGCAGGTTCTCTTTCGAGCACGAATCCATCTCTGGA AACTTCCTGCCATGTCAAAGGTG GGTAAGGTTTTTCGCGTTGCATCGAATTAAACCACATCATCCACCG CTTGTGCGGGTCCCCGTCAATT CCTTTGAGTTTCAACCTTGCGGCCGTACTCCCCAGGCGGTCAACT TCACGCGTTAGCTTCGTTACTGA GAAAACTAATTCCCAACAACCAGTTGACATCGTTTAGGGCGTGGA CTACCAGGGTATCTAATCCTGTT TGCTCCCCACGCTTTCGTGCATGAGCGTCAGTACAGGTCCAGGG GATTGCCTTCGCCATCGGTGTTC CTCCGCATATCTACGCATTTCACTGCTACACGCGGAATTCCATCC CCCTCTACCGTACTCTAGCCATG CAGTCACAAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACAT CTGTCTTACATAACCGCCTGCGC ACGCTTTACGCCCAGTAATTCCGATTAACGCTCGCACCCTACGTAT TACCGCGGCTGCTGGCACGTAG TTAGCCGGTGCTTATTCTTACGGTACCGTCATGGGCCCCCTGTATT AGAAGGAGCTTTTTCGTTCCGTA CAAAAGCAGTTTACAACCCGAAGGCCTTCATCCTGCACGCGGCAT TGCTGGATCAGGCTTTCGCCCA TTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCG TGTCTCAGTCCCAGTGTGGCTG GTCGTCCTCTCAGACCAGCTACAGATCGTCGGCTTGGTAAGCTTT TATCCCACCAACTACCTAATCTG CCATCGGCCGCTCCAATCGCGCGAGGCCCGAAGGGCCCCCGCTT TCATCCTCAGATCGTATGCGGTA TTAGCTACTCTTTCGAGTAGTTATCCCCCACGACTGGGCACGTTC CGATGTATTACTCACCCGTTCGC CACTCGTCAGCGTCCGAAGACCTGTTACCGTTCGACTTGCATGTG TAAGGCATGCCGCCAGCGTTCA ATCTGAGCCAGGATCAAACTCTACAGTTCGATCT (SEQ ID NO: 38) Pelomonas Bombyx mor GutATCCTGGCTCAGATTGAACGCT GGCGGCATGCCTTACACATGCA AGTCGAACGGTAACAGGTTAAGCTGACGAGTGGCGAACGGGTGA GTAATATATCGGAACGTGCCCA GTCGTGGGGGATAACTGCTCGAAAGAGCAGCTAATACCGCATAC GACCTGAGGGTGAAAGCGGGG GATCGCAAGACCTCGCNNGATTGGAGCGGCCGATATCAGATTAG GTAGTTGGTGGGGTAAAGGCCC ACCAAGCCAACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCA CACTGGGACTGAGACACGGCCC AGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGC GCAAGCCTGATCCAGCCATGCC GCGTGCGGGAAGAAGGCCTTCGGGTTGTAAACCGCTTTTGTCAGG GAAGAAAAGGTTCTGGTTAATAC CTGGGACTCATGACGGTACCTGAAGAATAAGCACCGGCTAACTAC GTGCCAGCAGCCGCGGTAATAC GTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGC GCAGGCGGTTATGCAAGACAGA GGTGAAATCCCCGGGCTCAACCTGGGAACTGCCTTTGTGACTGC ATAGCTAGAGTACGGTAGAGGG GGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGA GGAACACCGATGGCGAAGGCAA TCCCCTGGACCTGTACTGACGCTCATGCACGAAAGCGTGGGGAG CAAACAGGATTAGATACCCTGGT AGTCCACGCCCTAAACGATGTCAACTGGTTGTTGGGAGGGTTTCT TCTCAGTAACGTANNTAACGCGT GAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAA AGGAATTGACGGGGACCCGCAC AAGCGGTGGATGATGTGGTTTAATTCGATGCAACGCGAAAAACCT TACCTACCCTTGACATGCCAGGA ATCCTGAAGAGATTTGGGAGTGCTCGAAAGAGAACCTGGACACA GGTGCTGCATGGCCGTCGTCAG CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC CCTTGTCATTAGTTGCTACGAAA GGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGG GGATGACGTCAGGTCATCATGG CCCTTATGGGTAGGGCTACACACGTCATACAATGGCCGGGACAG AGGGCTGCCAACCCGCGAGGG GGAGCTAATCCCAGAAACCCGGTCGTAGTCCGGATCGTAGTCTG CAACTCGACTGCGTGAAGTCGG AATCGCTAGTAATCGCGGATCAGCTTGCCGCGGTGAATACGTTC CCGGGTCTTGTACACACCGCCC GTCACACCATGGGAGCGGGTTCTGCCAGAAGTAGTTAGCCTAACC GCAAGGAGGGCGATTACCACGG CAGGGTTCGTGACTGGGGTGAAGTCGTAACAAGGTAGCCGTATC GGAAGGTGCGGCTGGATCAC (SEQ ID NO: 39)

For example, a mosquito (e.g., Aedes spp. or Anopheles spp.) harborssymbiotic bacteria that modulate the mosquito's immune response andinfluence vectorial competence to pathogens. The modulating agentdescribed herein may be useful in targeting bacteria resident in themosquito, including, but not limited to, EspZ, Seratia spp (e.g.,Serratia marcescens), Enterbacterioaceae spp., Enterobacter spp. (e.g.,Enterobacter cloacae, Enterobacter amnigenus, Enterobacter ludwigii), .Proteus spp., Acinetobacter spp., Wigglesworthia spp. (Wigglesworthiagloosinidia), Xanthomonas spp. (e.g., Xanthomonas maltophilia),Pseudomonas spp. (e.g., Pseudomonas aeruginosa, Pseudomonas stutzeri,Pseudomonas rhodesiae), Escherichia spp. (e.g., Escherchia coli),Cedecea spp. (e.g., Cedecea lapagei), Ewingella spp. (e.g., Ewingellaamericana), Bacillus spp. (e.g., Bacillus pumilus), Comamonas spp., orVagococcus spp. (e.g., Vagococcus salmoninarium), or Wolbachia spp.(e.g., Wolbachia-wMel, Wolbachia-wAlbB, Wolbachia-wMelPop,Wolbachia-wMelPop-CLA).

Any number of bacterial species may be targeted by the compositions ormethods described herein. For example, in some instances, the modulatingagent may target a single bacterial species. In some instances, themodulating agent may target at least about any of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500,or more distinct bacterial species. In some instances, the modulatingagent may target any one of about 1 to about 5, about 5 to about 10,about 10 to about 20, about 20 to about 50, about 50 to about 100, about100 to about 200, about 200 to about 500, about 10 to about 50, about 5to about 20, or about 10 to about 100 distinct bacterial species. Insome instances, the modulating agent may target at least about any of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, ormore phyla, classes, orders, families, or genera of bacteria.

In some instances, the modulating agent may increase a population of oneor more bacteria (e.g., pathogenic bacteria, toxin-producing bacteria)by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ormore in the host in comparison to a host organism to which themodulating agent has not been administered. In some instances, themodulating agent may reduce the population of one or more bacteria(e.g., symbiotic bacteria, a pesticide-degrading bacterium, e.g., abacterium that degrades a pesticide listed in Table 12) by at leastabout any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in thehost in comparison to a host organism to which the modulating agent hasnot been administered. In some instances, the modulating agent mayeradicate the population of a bacterium (e.g., symbiotic bacteria, apesticide-degrading bacterium) in the host. In some instances, themodulating agent may increase the level of one or more pathogenicbacteria by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or more in the host and/or decreases the level of one or moresymbiotic bacteria by at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or more in the host in comparison to a host organismto which the modulating agent has not been administered.

In some instances, the modulating agent may alter the bacterialdiversity and/or bacterial composition of the host. In some instances,the modulating agent may increase the bacterial diversity in the hostrelative to a starting diversity by at least about any of 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or more in comparison to a host organismto which the modulating agent has not been administered. In someinstances, the modulating agent may decrease the bacterial diversity inthe host relative to a starting diversity by at least about any of 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more in comparison to a hostorganism to which the modulating agent has not been administered.

In some instances, the modulating agent may alter the function,activity, growth, and/or division of one or more bacterial cells. Forexample, the modulating agent may alter the expression of one or genesin the bacteria. In some instances, the modulating agent may alter thefunction of one or more proteins in the bacteria. In some instances, themodulating agent may alter the function of one or more cellularstructures (e.g., the cell wall, the outer or inner membrane) in thebacteria. In some instances, the modulating agent may kill (e.g., lyse)the bacteria.

The target bacterium may reside in one or more parts of the insect.Further, the target bacteria may be intracellular or extracellular. Insome instances, the bacteria reside in or on one or more parts of thehost gut, including, e.g., the foregut, midgut, and/or hindgut. In someinstances, the bacteria reside as an intracellular bacteria within acell of the host insect. In some instances, the bacteria reside in abacteriocyte of the host insect.

Changes to the populations of bacteria in the host may be determined byany methods known in the art, such as microarray, polymerase chainreaction (PCR), real-time PCR, flow cytometry, fluorescence microscopy,transmission electron microscopy, fluorescence in situ hybridization(e.g., FISH), spectrophotometry, matrix-assisted laser desorptionionization-mass spectrometry (MALDI-MS), and DNA sequencing. In someinstances, a sample of the host treated with a modulating agent issequenced (e.g., by metagenomics sequencing of 16S rRNA or rDNA) todetermine the microbiome of the host after delivery or administration ofthe modulating agent. In some instances, a sample of a host that did notreceive the modulating agent is also sequenced to provide a reference.

ii. Fungi

Exemplary fungi that may be targeted in accordance with the methods andcompositions provided herein, include, but are not limited toAmylostereum areolatum, Epichloe spp, Pichia pinus, Hansenula capsulate,Daldinia decipien, Ceratocytis spp, Ophiostoma spp, and Attamycesbromatificus. Non-limiting examples of yeast and yeast-like symbiontsfound in insects include Candida, Metschnikowia, Debaromyces,Scheffersomyces shehatae and Scheffersomyces stipites, Starmerella,Pichia, Trichosporon, Cryptococcus, Pseudozyma, and yeast-like symbiontsfrom the subphylum Pezizomycotina (e.g., Symbiotaphrina bucneri andSymbiotaphrina kochii). Non-limiting examples of yeast that may betargeted by the methods and compositions herein are listed in Table 2.

TABLE 2 Insect Species Order: Family Yeast Location (Species) Stegobiumpaniceum Coleoptera: Anobiidae Mycetomes (= Sitodrepa panicea)(Saccharomyces) Cecae (Torulopsis buchnerii) Mycetome between foregutand midgut Mycetomes (Symbiotaphrina buchnerii) Mycetomes and digestivetube (Torulopsis buchnerii) Gut cecae (Symbiotaphrina buchnerii)Lasioderma serricome Coleoptera: Anobiidae Mycetome between foregut andmidgut (Symbiotaphrina kochii) Emobius abietis Coleoptera: AnobiidaeMycetomes (Torulopsis karawaiewii) (Candida karawaiewii) Emobius mollisColeoptera: Anobiidae Mycetomes (Torulopsis emobii) (Candida emobii)Hemicoelus gibbicollis Coleoptera: Anobiidae Larval mycetomes Xestobiumplumbeum Coleoptera: Anobiidae Mycetomes (Torulopsis xestobii) (Candidaxestobii) Criocephalus rusticus Coleoptera: Cerambycidae MycetomesPhoracantha Coleoptera: Cerambycidae Alimentary canal (Candidasemipunctata guilliermondii, C. tenuis) Cecae around midgut (Candidaguilliermondii) Harpium inquisitor Coleoptera: Cerambycidae Mycetomes(Candida rhagii) Harpium mordax Coleoptera: Cerambycidae Cecae aroundmidgut (Candida tenuis) H. sycophanta Gaurotes virginea Coleoptera:Cerambycidae Cecae around midgut (Candida rhagii) Leptura rubraColeoptera: Cerambycidae Cecae around midgut (Candida tenuis) Cecaearound midgut (Candida parapsilosis) Leptura maculicomis Coleoptera:Cerambycidae Cecae around midgut (Candida parapsilosis) L.cerambyciformis Leptura sanguinolenta Coleoptera: Cerambycidae Cecaearound midgut (Candida sp.) Rhagium bifasciatum Coleoptera: CerambycidaeCecae around midgut (Candida tenuis) Rhagium inquisitor Coleoptera:Cerambycidae Cecae around midgut (Candida guilliermondii) Rhagium mordaxColeoptera: Cerambycidae Cecae around midgut (Candida) CarpophilusColeoptera: Nitidulidae Intestinal tract (10 yeast species) hemipterusOdontotaenius Coleoptera: Passalidae Hindgut (Enteroramus dimorphus)disjunctus Odontotaenius Coleoptera: Passalidae Gut (Pichia stipitis, P.segobiensis, disjunctus Candida shehatae) Verres stembergianus (C.ergatensis) Scarabaeus Coleoptera: Scarabaeidae Digestive tract (10yeast species) semipunctatus Chironitis furcifer Unknown speciesColeoptera: Scarabaeidae Guts (Trichosporon cutaneum) Dendroctonus andIps Coleoptera: Scolytidae Alimentary canal (13 yeast species) spp.Dendroctonus frontalis Coleoptera: Scolytidae Midgut (Candida sp.) Ipssexdentatus Coleoptera: Scolytidae Digestive tract (Pichia bovis, P.rhodanensis) Hansenula holstii (Candida rhagii) Digestive tract (Candidapulcherina) Ips typographus Coleoptera: Scolytidae Alimentary canalAlimentary tracts (Hansenula capsulata, Candida parapsilosis) Guts andbeetle homogenates (Hansenula holstii, H. capsulata, Candida diddensii,C. mohschtana, C. nitratophila, Cryptococcus albidus, C. laurentii)Trypodendron Coleoptera: Scolytidae Not specified lineatum Xyloterinuspolitus Coleoptera: Scolytidae Head, thorax, abdomen (Candida, Pichia,Saccharomycopsis) Periplaneta americana Dictyoptera: Blattidae Hemocoel(Candida sp. nov.) Blatta orientalis Dictyoptera: Blattidae Intestinaltract (Kluyveromyces blattae) Blatella germanica Dictyoptera:Blattellidae Hemocoel Cryptocercus Dictyoptera: Cryptocercidae Hindgut(1 yeast species) punctulatus Philophylla heraclei Diptera: TephritidaeHemocoel Aedes (4 species) Diptera: Culicidae Internal microflora (9yeast genera) Drosophila Diptera: Drosophilidae Alimentary canal (24yeast species) pseudoobscura Drosophila (5 spp.) Diptera: DrosophilidaeCrop (42 yeast species) Drosophila Diptera: Drosophilidae Crop (8 yeastspecies) melanogaster Drosophila (4 spp.) Diptera: Drosophilidae Crop (7yeast species) Drosophila (6 spp.) Diptera: Drosophilidae Larval gut (17yeast species) Drosophila (20 spp.) Diptera: Drosophilidae Crop (20yeast species) Drosophila (8 species Diptera: Drosophilidae Crop(Kloeckera, Candida, groups) Kluyveromyces) Drosophila serido Diptera:Drosophilidae Crop (18 yeast species) Drosophila (6 spp.) Diptera:Drosophilidae Intestinal epithelium (Coccidiascus legeri) ProtaxymiaDiptera Unknown (Candida, Cryptococcus, melanoptera Sporoblomyces)Astegopteryx styraci Homoptera: Aphididae Hemocoel and fat bodyTuberaphis sp. Homoptera: Aphididae Tissue sections Hamiltonaphisstyraci Glyphinaphis bambusae Cerataphis sp. Hamiltonaphis styraciHomoptera: Aphididae Abdominal hemocoel Cofana unimaculata Homoptera:Cicadellidae Fat body Leofa unicolor Homoptera: Cicadellidae Fat bodyLecaniines, etc. Homoptera: Coccoidea d Hemolymph, fatty tissue, etc.Lecanium sp. Homoptera: Coccidae Hemolymph, adipose tissue Ceroplastes(4 sp.) Homoptera: Coccidae Blood smears Laodelphax striatellusHomoptera: Delphacidae Fat body Eggs Eggs (Candida) Nilaparvata lugensHomoptera: Delphacidae Fat body Eggs (2 unidentified yeast species)Eggs, nymphs (Candida) Eggs (7 unidentified yeast species) Eggs(Candida) Nisia nervosa Homoptera: Delphacidae Fat body Nisia grandicepsPerkinsiella spp. Sardia rostrata Sogatella furcifera Sogatodesorizicola Homoptera: Delphacidae Fat body Amrasca devastans Homoptera:Jassidae Eggs, mycetomes, hemolymph Tachardina lobata Homoptera:Kerriidae Blood smears (Torulopsis) Laccifer (=Lakshadia) Homoptera:Kerriidae Blood smears (Torula variabilis) sp. Comperia mercetiHymenoptera: Encyrtidae Hemolymph, gut, poison gland Solenopsis invictaHymenoptera: Form icidae Hemolymph (Myrmecomyces annellisae) S.quinquecuspis Solenopsis invicta Hymenoptera: Form icidae Fourth instarlarvae (Candida parapsilosis, Yarrowia lipolytica) Gut and hemolymph(Candida parapsilosis, C. lipolytica, C. guillermondii, C. rugosa,Debaryomyces hansenii) Apis mellifera Hymenoptera: Apidae Digestivetracts (Torulopsis sp.) Intestinal tract (Torulopsis apicola) Digestivetracts (8 yeast species) Intestinal contents (12 yeast species)Intestinal contents (7 yeast species) Intestines (14 yeast species)Intestinal tract (Pichia melissophila) Intestinal tracts (7 yeastspecies) Alimentary canal (Hansenula silvicola) Crop and gut (13 yeastspecies) Apis mellifera Hymenoptera: Apidae Midguts (9 yeast genera)Anthophora Hymenoptera:Anthophoridae occidentalis Nomia melanderiHymenoptera:Halictidae Halictus rubicundus Hymenoptera:HalictidaeMegachile rotundata Hymenoptera:Megachilidae

Any number of fungal species may be targeted by the compositions ormethods described herein. For example, in some instances, the modulatingagent may target a single fungal species. In some instances, themodulating agent may target at least about any of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500,or more distinct fungal species. In some instances, the modulating agentmay target any one of about 1 to about 5, about 5 to about 10, about 10to about 20, about 20 to about 50, about 50 to about 100, about 100 toabout 200, about 200 to about 500, about 10 to about 50, about 5 toabout 20, or about 10 to about 100 distinct fungal species. In someinstances, the modulating agent may target at least about any of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, ormore phyla, classes, orders, families, or genera of fungi.

In some instances, the modulating agent may increase a population of oneor more fungi (e.g., pathogenic or parasitic fungi) by at least aboutany of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the hostin comparison to a host organism to which the modulating agent has notbeen administered. In some instances, the modulating agent may reducethe population of one or more fungi (e.g., symbiotic fungi) by at leastabout any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in thehost in comparison to a host organism to which the modulating agent hasnot been administered. In some instances, the modulating agent mayeradicate the population of a fungi (e.g., symbiotic fungi) in the host.In some instances, the modulating agent may increase the level of one ormore symbiotic fungi by at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or more in the host and/or may decrease the level ofone or more symbiotic fungi by at least about any of 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or more in the host in comparison to a hostorganism to which the modulating agent has not been administered.

In some instances, the modulating agent may alter the fungal diversityand/or fungal composition of the host. In some instances, the modulatingagent may increase the fungal diversity in the host relative to astarting diversity by at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or more in comparison to a host organism to whichthe modulating agent has not been administered. In some instances, themodulating agent may decrease the fungal diversity in the host relativeto a starting diversity by at least about any of 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or more in comparison to a host organism towhich the modulating agent has not been administered.

In some instances, the modulating agent may alter the function,activity, growth, and/or division of one or more fungi. For example, themodulating agent may alter the expression of one or more genes in thefungus. In some instances, the modulating agent may alter the functionof one or more proteins in the fungus. In some instances, the modulatingagent may alter the function of one or more cellular components in thefungus. In some instances, the modulating agent may kill the fungus.

Further, the target fungus may reside in one or more parts of theinsect. In some instances, the fungus resides in or on one or more partsof the insect gut, including, e.g., the foregut, midgut, and/or hindgut.In some instances, the fungus lives extracellularly in the hemolymph,fat bodies or in specialized structures in the host.

Changes to the population of fungi in the host may be determined by anymethods known in the art, such as microarray, polymerase chain reaction(PCR), real-time PCR, flow cytometry, fluorescence microscopy,transmission electron microscopy, fluorescence in situ hybridization(e.g., FISH), spectrophotometry, matrix-assisted laser desorptionionization-mass spectrometry (MALDI-MS), and DNA sequencing. In someinstances, a sample of the host treated with a modulating agent issequenced (e.g., by metagenomics sequencing) to determine the microbiomeof the host after delivery or administration of the modulating agent. Insome instances, a sample of a host that did not receive the modulatingagent is also sequenced to provide a reference.

III. Modulating Agents

The modulating agent of the methods and compositions provided herein mayinclude a phage, a polypeptide, a small molecule, an antibiotic, asecondary metabolite, a bacterium, a fungus, or any combination thereof.

i. Phage

The modulating agent described herein may include a phage (e.g., a lyticphage or a non-lytic phage). In some instances, an effectiveconcentration of any phage described herein may alter a level, activity,or metabolism of one or more microorganisms (as described herein)resident in a host described herein (e.g., a vector of a human pathogen,e.g., a mosquito, a mite, a biting louse, or a tick), the modulationresulting in a decrease in the host's fitness (e.g., as outlinedherein). In some instances, the modulating agent includes at least onephage selected from the order Tectiviridae, Myoviridae, Siphoviridae,Podoviridae, Caudovirales, Lipothrixviridae, Rudiviridae, orLigamenvirales. In some instances, the composition includes at least onephage selected from the family Myoviridae, Siphoviridae, Podoviridae,Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae,Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae,Gluboloviridae, Guttaviridae, Inoviridae, Leviviridae, Microviridae,Plasmaviridae, and Tectiviridae. Further non-limiting examples of phagesuseful in the methods and compositions are listed in Table 3.

TABLE 3 Examples of Phage and Targeted Bacteria Phage and Accession #Target bacteria Target host SA1 (NC_027991), phiP68 StaphylococcusApidae family (NC_004679) sp. WO (AB036666.1) Wolbachia sp. Aedesaegypt; Drosophila melanogaster; Plasmodium sp; Teleogryllus taiwanemma;Bombyx mori KL1 (NC_018278), BcepNazgul Burkholderia sp. Riptortus sp.;(NC_005091) Phi E125 Pyrrhocoris apterus. (NC_003309) Fern (NC_028851),Xenia Paenibacillus bumble bees: Bombus (NC_028837), HB10c2 larvae sp.;honey bees: A. (NC_028758) mellifera CP2 (NC_020205), XP10 XanthomonasPlebeina denoiti; (NC_004902), sp. Apidae family; XP15 (NC_007024),phiL7 Apis mellifera; (NC_012742) Drosphilidae family; and Chloropidaefamily PP1 (NC_019542), PM1 Pectobacterium Apidae family (NC_023865)carotovorum subsp. carotovorum φRSA1 (NC_009382), Ralstonia Bombyx moriφRSB1 (NC_011201), φRSL1 solanacearum (NC_010811), RSM1 (NC_008574)SF1(NC_028807) Streptomyces Philantus sp.; scabies Trachypus sp ECML-4(NC)_025446), Escherichia coli Apidae family; ECML-117 (NC_025441),Varroa destructor ECML-134 (NC_025449) SSP5(JX274646.1), SSP6 Salmonellasp. Drosphilidae family (NC_004831), SFP10 (NC_016073), F18SE(NC_028698) y (NC_001416), Bcp1 Bacillus sp. Gypsy moth; (NC_024137)Lymantria dispar; Varroa destructor Phil (NC_009821) EnterococcusSchistocerca gragaria sp. φKMV (NC 005045), Pseudomonas Lymantriadispar; φEL(AJ697969.1), φKZ sp. Apidae family (NC 004629) A2(NC_004112), phig1e Lactobacilli sp. Apidae family; (NC_004305)Drosophila family; Varroa destructor KLPN1 (NC_028760) Klebsiella sp. C.capitata vB_AbaM_Acibe1004 Acinetobacter Schistocerca gragaria(NC_025462), vB_AbaP_Acibe1007 sp. (NC_025457)

In some instances, a modulating agent includes a lytic phage. Thus,after delivery of the lytic phage to a bacterial cell resident in thehost, the phage causes lysis in the target bacterial cell. In someinstances, the lytic phage targets and kills a bacterium resident in ahost insect to decrease the fitness of the host. Alternatively oradditionally, the phage of the modulating agent may be a non-lytic phage(also referred to as lysogenic or temperate phage). Thus, after deliveryof the non-lytic phage to a bacterial cell resident in the host, thebacterial cell may remain viable and able to stably maintain expressionof genes encoded in the phage genome. In some instances, a non-lyticphage is used to alter gene expression in a bacterium resident in a hostinsect to decrease the fitness of the host. In some instances, themodulating agent includes a mixture of lytic and non-lytic phage.

In certain instances, the phage is a naturally occurring phage. Forexample, a naturally occurring phage may be isolated from anenvironmental sample containing a mixture of different phages. Thenaturally occurring phage may be isolated using methods known in the artto isolate, purify, and identify phage that target a particularmicroorganism (e.g., a bacterial endosymbiont in an insect host).Alternatively, in certain instances, the phage may be engineered basedon a naturally occurring phage.

The modulating agent described herein may include phage with either anarrow or broad bacterial target range. In some instances, the phage hasa narrow bacterial target range. In some instances, the phage is apromiscuous phage with a large bacterial target range. For example, thepromiscuous phage may target at least about any of 5, 10, 20, 30, 40,50, or more bacterium resident in the host. A phage with a narrowbacterial target range may target a specific bacterial strain in thehost without affecting another, e.g., non-targeted, bacterium in thehost. For example, the phage may target no more than about any of 50,40, 30, 20, 10, 8, 6, 4, 2, or 1 bacterium resident in the host. Forexample, the phage described herein may be useful in targeting one ormore bacteria resident in the mosquito, including, but not limited to,EspZ, Seratia spp (e.g., Serratia marcescens), Enterbacterioaceae spp.,Enterobacter spp. (e.g., Enterobacter cloacae, Enterobacter amnigenus,Enterobacter ludwigii), . Proteus spp., Acinetobacter spp.,Wigglesworthia spp. (Wigglesworthia gloosinidia), Xanthomonas spp.(e.g., Xanthomonas maltophilia), Pseudomonas spp. (e.g., Pseudomonasaeruginosa, Pseudomonas stutzeri, Pseudomonas rhodesiae), Escherichiaspp. (e.g., Escherchia coli), Cedecea spp. (e.g., Cedecea lapagel),Ewingella spp. (e.g., Ewingella americana), Bacillus spp. (e.g.,Bacillus pumilus), Comamonas spp., or Vagococcus spp. (e.g., Vagococcussalmoninarium), or Wolbachia spp. (e.g., Wolbachia-wMel,Wolbachia-wAlbB, Wolbachia-wMelPop, Wolbachia-wMelPop-CLA).

The compositions described herein may include any number of phage, suchas at least about any one of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100,or more phage. In some instances, the composition includes phage fromone or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phage) families,one or more orders (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phage), orone or more species (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100,or more phage). Compositions including one or more phage are alsoreferred herein as “phage cocktails.” Phage cocktails are useful becausethey allow for targeting of a wider host range of bacteria. Furthermore,they allow for each bacterial strain (i.e. subspecies) to be targeted bymultiple orthogonal phages, thereby preventing or significantly delayingthe onset of resistance. In some instances, a cocktail includes multiplephages targeting one bacterial species. In some instances, a cocktailincludes multiple phages targeting multiple bacterial species. In someinstances, a one-phage “cocktail” includes a single promiscuous phage(i.e. a phage with a large host range) targeting many strains within aspecies.

Suitable concentrations of the phage in the modulating agent describedherein depends on factors such as efficacy, survival rate,transmissibility of the phage, number of distinct phage, and/or lysintypes in the compositions, formulation, and methods of application ofthe composition. In some instances, the phage is in a liquid or a solidformulation. In some instances, the concentration of each phage in anyof the compositions described herein is at least about any of 10², 10³,10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ or more pfu/ml. In some instances,the concentration of each phage in any of the compositions describedherein is no more than about any of 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸,10⁹ pfu/ml. In some instances, the concentration of each phage in thecomposition is any of about 10² to about 10³, about 10³ to about 10⁴,about 10⁴ to about 10⁵, about 10⁵ to about 10⁶, about 10⁷ to about 10⁸,about 10⁸ to about 10⁹, about 10² to about 10⁴, about 10⁴ to about 10⁶,about 10⁶ to about 10⁹, or about 10³ to about 10⁸ pfu/ml. In someinstances, wherein the composition includes at least two types ofphages, the concentration of each type of the phages may be the same ordifferent. For example, in some instances, the concentration of onephage in the cocktail is about 10⁸ pfu/ml and the concentration of asecond phage in the cocktail is about 10⁶ pfu/ml.

A modulating agent including a phage as described herein can becontacted with the target host in an amount and for a time sufficientto: (a) reach a target level (e.g., a predetermined or threshold level)of phage concentration inside a target host; (b) reach a target level(e.g., a predetermined or threshold level) of phage concentration insidea target host gut; (c) reach a target level (e.g., a predetermined orthreshold level) of phage concentration inside a target hostbacteriocyte; (d) modulate the level, or an activity, of one or moremicroorganism (e.g., endosymbiont) in the target host; or/and (e)modulate fitness of the target host.

As illustrated by Examples 5-7 and 28, phages (e.g., one or morenaturally occurring phage) can be used as modulating agents that targetan endosymbiotic bacterium in an insect host to decrease the fitness ofthe host (e.g., as outlined herein).

ii. Polypeptides

Numerous polypeptides (e.g., a bacteriocin, R-type bacteriocin, noduleC-rich peptide, antimicrobial peptide, lysin, or bacteriocyte regulatorypeptide) may be used in the compositions and methods described herein.In some instances, an effective concentration of any peptide orpolypeptide described herein may alter a level, activity, or metabolismof one or more microorganisms (as described herein, e.g., a Wolbachiaspp. or a Rickettsia spp.) resident in a host (e.g., a vector of a humanpathogen, e.g., a mosquito, mite, biting louse, or tick), the modulationresulting in a decrease in the host's fitness (e.g., as outlinedherein). Polypeptides included herein may include naturally occurringpolypeptides or recombinantly produced variants. For example, thepolypeptide may be a functionally active variant of any of thepolypeptides described herein with at least 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g.,over a specified region or over the entire sequence, to a sequence of apolypeptide described herein or a naturally occurring polypeptide.

A modulating agent comprising a polypeptide as described herein can becontacted with the target host in an amount and for a time sufficientto: (a) reach a target level (e.g., a predetermined or threshold level)of concentration inside a target host; (b) reach a target level (e.g., apredetermined or threshold level) of concentration inside a target hostgut; (c) reach a target level (e.g., a predetermined or threshold level)of concentration inside a target host bacteriocyte; (d) modulate thelevel, or an activity, of one or more microorganism (e.g., endosymbiont)in the target host; or/and (e) modulate fitness of the target host.

The polypeptide modulating agents discussed hereinafter, namelybacteriocins, lysins, antimicrobial peptides, nodule C-rich peptides,and bacteriocyte regulatory peptides, can be used to alter the level,activity, or metabolism of target microorganisms (e.g., Rickettsia orWolbochia) as indicated in the section for decreasing the fitness ofhost insects (e.g., a vector of a human pathogen, e.g., a mosquito, amite, a biting louse, or a tick).

(a) Bacteriocins

The modulating agent described herein may include a bacteriocin. In someinstances, the bacteriocin is naturally produced by Gram-positivebacteria, such as Pseudomonas, Streptomyces, Bacillus, Staphylococcus,or lactic acid bacteria (LAB, such as Lactococcus lactis). In someinstances, the bacteriocin is naturally produced by Gram-negativebacteria, such as Hafnia alvei, Citrobacter freundii, Klebsiellaoxytoca, Klebsiella pneumonia, Enterobacter cloacae, Serratiaplymithicum, Xanthomonas campestris, Erwinia carotovora, Ralstoniasolanacearum, or Escherichia coli. Exemplary bacteriocins include, butare not limited to, Class I-IV LAB antibiotics (such as lantibiotics),colicins, microcins, and pyocins. Non-limiting examples of bacteriocinsare listed in Table 4.

TABLE 4 Examples of Bacteriocins Class Name Producer Targets SequenceClass I Nisin Lactococcus Active on Gram- ITSISLCTPGCKTGALMGCN lactispositive bacteria: MKTATCHCSIHVSK Enterococcus, (SEQ ID NO: 40)Lactobacillus, Lactococcus, Leuconostoc, Listeria, Clostridium EpiderminStaphylococcus IASKFICTPGCAKTGSFNSY epidermis CC (SEQ ID NO: 41)Class II Class II a Pediocin Pediococcus Pediococci,KYYGNGVTCGKHSCSCDWGK PA-1 acidilactici Lactobacilli,ATTCIINNGAMAWATGGHQG Leuconostoc, NHKC Brochothrix (SEQ ID NO: 42)thermosphacta, Propionibacteria, Bacilli, Enterococci, Staphylococci,Listeria clostridia, Listeria monocytogenes, Listeria innocua Class II bEnterocin P Enterococcus Lactobacillus sakei, ATRSYGNGVYCNNSKCWVNWfaecium Enterococcus faecium GEAKENIAGIVISGWASGLA GMGH (SEQ ID NO: 43)Class II c lactococcin G Streptococcus Gram-positive bacteriaGTWDDIGQGIGRVAYWVGKA lactis MGNMSDVNQASRINRKKKH (SEQ ID NO: 44)Class II d Lactacin-F Lactobacillus Lactobacilli, NRWGDTVLSAASGAGTGIKAjohnsonii Enterococcus faecalis CKSFGPWGMAICGVGGAAIG GYFGYTHN(SEQ ID NO: 45) Class III Class III a Enterocin EnterococcusBroad spectrum: Gram MAKEFGIPAAVAGTVLNVVE AS-48 faecalispositive and Gram AGGWVTTIVSILTAVGSGGL negative bacteria.SLLAAAGRESIKAYLKKEIK KKGKRAVIAW (SEQ ID NO: 46) Class III b aureocin A70Staphylococcus Broad spectrum: Gram MSWLNFLKYIAKYGKKAVSA aureuspositive and Gram AWKYKGKVLEWLNVGPTLEW negative bacteria. VWQKLKKIAGL(SEQ ID NO: 47) Class IV Garvicin A Lactococcus Broad spectrum: GramIGGALGNALNGLGTWANMMN garvieae positive and Gram GGGFVNQWQVYANKGKINQYnegative bacteria. RPY (SEQ ID NO: 48) Unclassified Colicin VEscherichia coli Active against MRTLTLNELDSVSGGASGRDEscherichia coli (also IAMAIGTLSGQFVAGGIGAA closely relatedAGGVAGGAIYDYASTHKPNP bacteria); AMSPSGLGGTIKQKPEGIPS EnterbacteriaceaeEAWNYAAGRLCNWSPNNLSD VCL (SEQ ID NO: 49)

In some instances, the bacteriocin is a colicin, a pyocin, or a microcinproduced by Gram-negative bacteria. In some instances, the bacteriocinis a colicin. The colicin may be a group A colicin (e.g., uses the Tolsystem to penetrate the outer membrane of a target bacterium) or a groupB colicin (e.g., uses the Ton system to penetrate the outer membrane ofa target bacterium). In some instances, the bacteriocin is a microcin.The microcin may be a class I microcin (e.g., <5 kDa, haspost-translational modifications) or a class II microcin (e.g., 5-10kDa, with or without post-translational modifications). In someinstances, the class II microcin is a class IIa microcin (e.g., requiresmore than one genes to synthesize and assemble functional peptides) or aclass IIb microcin (e.g., linear peptides with or withoutpost-translational modifications at C-terminus). In some instances, thebacteriocin is a pyocin. In some instances, the pyocin is an R-pyocin,F-pyocin, or S-pyocin.

In some instances, the bacteriocin is a class I, class II, class III, orclass IV bacteriocin produced by Gram-positive bacteria. In someinstances, the modulating agent includes a Class I bacteriocin (e.g.,lanthionine-containing antibiotics (lantibiotics) produced by aGram-positive bacteria). The class I bacteriocins or lantibiotic may bea low molecular weight peptide (e.g., less than about 5 kDa) and maypossess post-translationally modified amino acid residues (e.g.,lanthionine, β-methyllanthionine, or dehydrated amino acids).

In some instances, the bacteriocin is a Class II bacteriocin (e.g.,non-lantibiotics produced by Gram-positive bacteria). Many arepositively charged, non-lanthionine-containing peptides, which unlikelantibiotics, do not undergo extensive post-translational modification.The Class II bacteriocin may belong to one of the following subclasses:“pediocin-like” bacteriocins (e.g., pediocin PA-1 and carnobacteriocin X(Class IIa)); two-peptide bacteriocins (e.g., lactacin F and ABP-118(Class IIb)); circular bacteriocins (e.g., carnocyclin A and enterocinAS-48 (Class 11c)); or unmodified, linear, non-pediocin-likebacteriocins (e.g., epidermicin NI01 and lactococcin A (Class IId)).

In some instances, the bacteriocin is a Class III bacteriocin (e.g.,produced by Gram-positive bacteria). Class III bacteriocins may have amolecular weight greater than 10 kDa and may be heat unstable proteins.The Class III bacteriocins can be further subdivided into Group IIIA andGroup IIIB bacteriocins. The Group IIIA bacteriocins includebacteriolytic enzymes which kill sensitive strains by lysis of the cellwell, such as Enterolisin A. Group IIIB bacteriocins include non-lyticproteins, such as Caseicin 80, Helveticin J, and lactacin B.

In some instances, the bacteriocin is a Class IV bacteriocin (e.g.,produced by Gram-positive bacteria). Class IV bacteriocins are a groupof complex proteins, associated with other lipid or carbohydratemoieties, which appear to be required for activity. They are relativelyhydrophobic and heat stable. Examples of Class IV bacteriocinsleuconocin S, lactocin 27, and lactocin S.

In some instances, the bacteriocin is an R-type bacteriocin. R-typebacteriocins are contractile bacteriocidal protein complexes. SomeR-type bacteriocins have a contractile phage-tail-like structure. TheC-terminal region of the phage tail fiber protein determinestarget-binding specificity. They may attach to target cells through areceptor-binding protein, e.g., a tail fiber. Attachment is followed bysheath contraction and insertion of the core through the envelope of thetarget bacterium. The core penetration results in a rapid depolarizationof the cell membrane potential and prompt cell death. Contact with asingle R-type bacteriocin particle can result in cell death. An R-typebacteriocin, for example, may be thermolabile, mild acid resistant,trypsin resistant, sedimentable by centrifugation, resolvable byelectron microscopy, or a combination thereof. Other R-type bacteriocinsmay be complex molecules including multiple proteins, polypeptides, orsubunits, and may resemble a tail structure of bacteriophages of themyoviridae family. In naturally occurring R-type bacteriocins, thesubunit structures may be encoded by a bacterial genome, such as that ofC. difficile or P. aeruginosa and form R-type bacteriocins to serve asnatural defenses against other bacteria. In some instances, the R-typebacteriocin is a pyocin. In some instances, the pyocin is an R-pyocin,F-pyocin, or S-pyocin.

In some instances, the bacteriocin is a functionally active variant ofthe bacteriocins described herein. In some instances, the variant of thebacteriocin has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specifiedregion or over the entire sequence, to a sequence of a bacteriocindescribed herein or a naturally occurring bacteriocin.

In some instances, the bacteriocin may be bioengineered, according tostandard methods, to modulate their bioactivity, e.g., increase ordecrease or regulate, or to specify their target microorganisms. Inother instances, the bacteriocin is produced by the translationalmachinery (e.g. a ribosome, etc.) of a microbial cell. In someinstances, the bacteriocin is chemically synthesized. Some bacteriocinscan be derived from a polypeptide precursor. The polypeptide precursorcan undergo cleavage (e.g., processing by a protease) to yield thepolypeptide of the bacteriocin itself. As such, in some instances, thebacteriocin is produced from a precursor polypeptide. In some otherinstances, the bacteriocin includes a polypeptide that has undergonepost-translational modifications, for example, cleavage, or the additionof one or more functional groups.

The bacteriocins described herein may be formulated in a composition forany of the uses described herein. The compositions disclosed herein mayinclude any number or type (e.g., classes) of bacteriocins, such as atleast about any one of 1 bacteriocin, 2, 3, 4, 5, 10, 15, 20, 30, 40,50, 100, or more bacteriocins. Suitable concentrations of eachbacteriocin in the compositions described herein depends on factors suchas efficacy, stability of the bacteriocin, number of distinctbacteriocin types in the compositions, formulation, and methods ofapplication of the composition. In some instances, each bacteriocin in aliquid composition is from about 0.01 ng/ml to about 100 mg/mL. In someinstances, each bacteriocin in a solid composition is from about 0.01ng/g to about 100 mg/g. In some instances, wherein the compositionincludes at least two types of bacteriocins, the concentration of eachtype of the bacteriocins may be the same or different. In someinstances, the bacteriocin is provided in a composition including abacterial cell that secretes the bacteriocin. In some instances, thebacteriocin is provided in a composition including a polypeptide (e.g.,a polypeptide isolated from a bacterial cell).

Bacteriocins may neutralize (e.g., kill) at least one microorganismother than the individual bacterial cell in which the polypeptide ismade, including cells clonally related to the bacterial cell and othermicrobial cells. As such, a bacterial cell may exert cytotoxic orgrowth-inhibiting effects on a plurality of microbial organisms bysecreting bacteriocins. In some instances, the bacteriocin targets andkills one or more species of bacteria resident in the host viacytoplasmic membrane pore formation, cell wall interference (e.g.,peptidoglycanase activity), or nuclease activity (e.g., DNase activity,16S rRNase activity, or tRNase activity).

In some instances, the bacteriocin has a neutralizing activity.Neutralizing activity of bacteriocins may include, but is not limitedto, arrest of microbial reproduction, or cytotoxicity. Some bacteriocinshave cytotoxic activity, and thus can kill microbial organisms, forexample bacteria, yeast, algae, and the like. Some bacteriocins caninhibit the reproduction of microbial organisms, for example bacteria,yeast, algae, and the like, for example by arresting the cell cycle.

In some instances, the bacteriocin has killing activity. The killingmechanism of bacteriocins is specific to each group of bacteriocins. Insome instances, the bacteriocin has narrow-spectrum bioactivity.Bacteriocins are known for their very high potency against their targetstrains. Some bacteriocin activity is limited to strains that areclosely related to the bacteriocin producer strain (narrow-spectrumbioactivity). In some instances, the bacteriocin has broad-spectrumbioactivity against a wide range of genera.

In some instances, bacteriocins interact with a receptor molecule or adocking molecule on the target bacterial cell membrane. For example,nisin is extremely potent against its target bacterial strains, showingantimicrobial activity even at a single-digit nanomolar concentration.The nisin molecule has been shown to bind to lipid II, which is the maintransporter of peptidoglycan subunits from the cytoplasm to the cellwall

In some instances, the bacteriocin has anti-fungal activity. A number ofbacteriocins with anti-yeast or anti-fungal activity have beenidentified. For example, bacteriocins from Bacillus have been shown tohave neutralizing activity against some yeast strains (see, for example,Adetunji and Olaoye, Malaysian Journal of Microbiology 9:130-13, 2013).In another example, an Enterococcus faecalis peptide has been shown tohave neutralizing activity against Candida species (see, for example,Shekh and Roy, BMC Microbiology 12:132, 2012). In another example,bacteriocins from Pseudomonas have been shown to have neutralizingactivity against fungi, such as Curvularia lunata, Fusarium species,Helminthosporium species, and Biopolaris species (see, for example,Shalani and Srivastava, The Internet Journal of Microbiology Volume 5Number 2, 2008). In another example, botrycidin AJ1316 and alirin B1from B. subtilis have been shown to have antifungal activities.

A modulating agent including a bacteriocin as described herein can becontacted with the target host in an amount and for a time sufficientto: (a) reach a target level (e.g., a predetermined or threshold level)of bacteriocin concentration inside a target host; (b) reach a targetlevel (e.g., a predetermined or threshold level) of bacteriocinconcentration inside a target host gut; (c) reach a target level (e.g.,a predetermined or threshold level) of bacteriocin concentration insidea target host bacteriocyte; (d) modulate the level, or an activity, ofone or more microorganism (e.g., endosymbiont) in the target host;or/and (e) modulate fitness of the target host.

As illustrated by Examples 8, 9, and 16, bacteriocins (e.g., colA ornisin) can be used as modulating agents that target an endosymbioticbacterium in an insect host to decrease the fitness of the host (e.g.,as outlined herein).

(b) Lysins

The modulating agent described herein may include a lysin (e.g., alsoknown as a murein hydrolase or peptidoglycan autolysin). Any lysinsuitable for inhibiting a bacterium resident in the host may be used. Insome instances, the lysin is one that can be naturally produced by abacterial cell. In some instances, the lysin is one that can benaturally produced by a bacteriophage. In some instances, the lysin isobtained from a phage that inhibits a bacterium resident in the host. Insome instances, the lysin is engineered based on a naturally occurringlysin. In some instances, the lysin is engineered to be secreted by ahost bacterium, for example, by introducing a signal peptide to thelysin. In some instances, the lysin is used in combination with a phageholin. In some instances, a lysin is expressed by a recombinantbacterium host that is not sensitive to the lysin. In some instances,the lysin is used to inhibit a Gram-positive or Gram-negative bacteriumresident in the host.

The lysin may be any class of lysin and may have one or more substratespecificities. For example, the lysin may be a glycosidase, anendopeptidase, a carboxypeptidase, or a combination thereof. In someinstances, the lysin cleaves the β-1-4 glycosidic bond in the sugarmoiety of the cell wall, the amide bond connecting the sugar and peptidemoieties of the bacterial cell wall, and/or the peptide bonds betweenthe peptide moieties of the cell wall. The lysin may belong to one ormore specific lysin groups, depending on the cleavage site within thepeptidoglycan. In some instances, the lysin is a N-acetyl-β-D-muramidase(e.g., lysozyme), lytic transglycosylase, N-acetyl-β-D-glucosaminidase,N-acetylmuramyl-L-alanine amidase, L,D-endopeptidase, D,D-endopeptidase,D,D-carboxypeptidase, L,D-carboxypeptidase, or L,D-transpeptidase.Non-limiting examples of lysins and their activities are listed in Table5.

TABLE 5 Examples of Lysins Target Bacteria Producer Lysins ActivitySequence S. pneumoniae Cp1 Cpl-1 Muramidase MVKKNDLFVDVSSHNGYDITGILEQMGTTNTIIKISESTTYLNPCLSAQV EQSNPIGFYHFARFGGDVAEAEREAQFFLDNVPMQVKYLVLDYEDDPSGD AQANTNACLRFMQMIADAGYKPIYYSYKPFTHDNVDYQQILAQFPNSLWI AGYGLNDGTANFEYFPSMDGIRWWQYSSNPFDKNIVLLDDEEDDKPKTAG TWKQDSKGWWFRRNNGSFPYNKWEKIGGVWYYFDSKGYCLTSEWLKDNEK WYYLKDNGAMATGWVLVGSEWYYMDDSGAMVTGWVKYKNNWYYMTNERGN MVSNEFIKSGKGWYFMNTNGELADN PSFTKEPDGLITVA(SEQ ID NO: 50) S. pneumoniae Dp-1 Pal Amidase MGVDIEKGVAWMQARKGRVSYSMDFRDGPDSYDCSSSMYYALRSAGASSA GWAVNTEYMHAWLIENGYELISENAPWDAKRGDIFIWGRKGASAGAGGHT GMFIDSDNIIHCNYAYDGISVNDHDERWYYAGQPYYYVYRLTNANAQPAE KKLGWQKDATGFWYARANGTYPKDEFEYIEENKSWFYFDDQGYMLAEKWL KHTDGNWYWFDRDGYMATSWKRIGESWYYFNRDGSMVTGWIKYYDNWYYC DATNGDMKSNAFIRYNDGWYLLLPDGRLADKPQFTVEPDGLITAKV (SEQ ID NO: 51) S. pyogenes C1 Cl Amidase N/AB. anthracis γ PlyG Amidase MEIQKKLVDPSKYGTKCPYTMKPKYITVHNTYNDAPAENEVSYMISNNNE VSFHIAVDDKKAIQGIPLERNAWACGDGNGSGNRQSISVEICYSKSGGDR YYKAEDNAVDVVRQLMSMYNIPIENVRTHQSWSGKYCPHRMLAEGRWGAF IQKVKNGNVATTSPTKQNIIQSGAFSPYETPDVMGALTSLKMTADFILQS DGLTYFISKPTSDAQLKAMKEYLDR KGWWYEVK(SEQ ID NO: 52) B. anthracis Ames prophage PlyPH Amidase N/AE. faecalis and Phi1 PlyV12 Amidase N/A E. faecium S. aureus ΦMR11 MV-LEndopeptidase and MQAKLTKKEFIEWLKTSEGKQFNVD amidaseLWYGFQCFDYANAGWKVLFGLLLKG LGAKDIPFANNFDGLATVYQNTPDFLAQPGDMVVFGSNYGAGYGHVAWVI EATLDYIIVYEQNWLGGGWTDRIEQPGWGWEKVTRRQHAYDFPMWFIRPN FKSETAPRSIQSPTQASKKETAKPQPKAVELKIIKDVVKGYDLPKRGGNP KGIVIHNDAGSKGATAEAYRNGLVNAPLSRLEAGIAHSYVSGNTVWQALD ESQVGWHTANQLGNKYYYGIEVCQSMGADNATFLKNEQATFQECARLLKK WGLPANRNTIRLHNEFTSTSCPHRSSVLHTGFDPVTRGLLPEDKQLQLKD YFIKQIRVYMDGKIPVATVSNESSASSNTVKPVASAWKRNKYGTYYMEEN ARFTNGNQPITVRKIGPFLSCPVAYQFQPGGYCDYTEVMLQDGHVWVGYT WEGQRYYLPIRTWNGSAPPNQILGD LWGEIS(SEQ ID NO: 53) S. pyogenes C1 PlyC Amidase N/A S. agalactiae B30GBS lysin Muramidase and MVINIEQAIAWMASRKGKVTYSMDY endopeptidaseRNGPSSYDCSSSVYFALRSAGASDN GWAVNTEYEHDWLIKNGYVLIAENTNWNAQRGDIFIWGKRGASAGAFGHT GMFVDPDNIIHCNYGYNSITVNNHDEIWGYNGQPYVYAYRYSGKQSNAKV DNKSVVSKFEKELDVNTPLSNSNMPYYEATISEDYYVESKPDVNSTDKEL LVAGTRVRVYEKVKGWARIGAPQSN QWVEDAYLIDATDM(SEQ ID NO: 54) S. aureus P68 Lys16 Endopeptidase N/A S. aureus K LysKAmidase and MAKTQAEINKRLDAYAKGTVDSPYR endopeptidaseVKKATSYDPSFGVMEAGAIDADGYY HAQCQDLITDYVLWLTDNKVRTWGNAKDQIKQSYGTGFKIHENKPSTVPK KGWIAVFTSGSYEQWGHIGIVYDGGNTSTFTILEQNWNGYANKKPTKRVD NYYGLTHFIEIPVKAGTTVKKETAKKSASKTPAPKKKATLKVSKNHINYT MDKRGKKPEGMVIHNDAGRSSGQQYENSLANAGYARYANGIAHYYGSEGY VWEAIDAKNQIAWHTGDGTGANSGNFRFAGIEVCQSMSASDAQFLKNEQA VFQFTAEKFKEWGLTPNRKTVRLHMEFVPTACPHRSMVLHTGFNPVTQGR PSQAIMNKLKDYFIKQIKNYMDKGTSSSTVVKDGKTSSASTPATRPVTGS WKKNQYGTWYKPENATFVNGNQPIVTRIGSPFLNAPVGGNLPAGATIVYD EVCIQAGHIWIGYNAYNGNRVYCPV RTCQGVPPNQIPGVAWGVFK(SEQ ID NO: 55) L. monocytogenes A118 Ply118 AmidaseMTSYYYSRSLANVNKLADNTKAAAR KLLDWSESNGIEVLIYETIRTKEQQAANVNSGASQTMRSYHLVGQALDFV MAKGKTVDWGAYRSDKGKKFVAKAKSLGFEWGGDWSGFVDNPHLQFNYKG YGTDTFGKGASTSNSSKPSADTNTNSLGLVDYMNLNKLDSSFANRKKLAT SYGIKNYSGTATQNTTLLAKLKAGKPHTPASKNTYYTENPRKVKTLVQCD LYKSVDFTTKNQTGGTFPPGTVFTISGMGKTKGGTPRLKTKSGYYLTANT KFVKKI (SEQ ID NO: 56) L. monocytogenes A511Ply511 Amidase MVKYTVENKIIAGLPKGKLKGANFV IAHETANSKSTIDNEVSYMTRNWKNAFVTHFVGGGGRVVQVANVNYVSWG AGQYANSYSYAQVELCRTSNATTFKKDYEVYCQLLVDLAKKAGIPITLDS GSKTSDKGIKSHKWVADKLGGTTHQDPYAYLSSWGISKAQFASDLAKVSG GGNTGTAPAKPSTPAPKPSTPSTNLDKLGLVDYMNAKKMDSSYSNRDKLA KQYGIANYSGTASQNTTLLSKIKGGAPKPSTPAPKPSTSTAKKIYFPPNK GNWSVYPTNKAPVKANAIGAINPTKFGGLTYTIQKDRGNGVYEIQTDQFG RVQVYGAPSTGAVIKK (SEQ ID NO: 57)L. monocytogenes A500 Ply500 Endopeptidase MALTEAWLIEKANRKLNAGGMYKITSDKTRNVIKKMAKEGIYLCVAQGYR STAEQNALYAQGRTKPGAIVTNAKGGQSNHNYGVAVDLCLYTNDGKDVIW ESTTSRWKKVVAAMKAEGFKWGGDWKSFKDYPHFELCDAVSGEKIPAATQ NTNTNSNRYEGKVIDSAPLLPKMDFKSSPFRMYKVGTEFLVYDHNQYWYK TYIDDKLYYMYKSFCDVVAKKDAKGRIKVRIKSAKDLRIPVWNNIKLNSG KIKWYAPNVKLAWYNYRRGYLELWY PNDGWYYTAEYFLK(SEQ ID NO: 58) S. pneumoniae ΦDp-1 Pal, S Endopeptidase and N/A amidaseS. agalactiae LambdaSa1 LambdaSa1 Glycosidase MVINIEQAIAWMASRKGKVTYSMDYprophage RNGPSSYDCSSSVYFALRSAGASDN GWAVNTEYEHDWLIKNGYVLIAENTNWNAQRGDIFIWGKRGASAGAFGHT GMFVDPDNIIHCNYGYNSITVNNHDEIWGYNGQPYVYAYRYARKQSNAKV DNQSVVSKFEKELDVNTPLSNSNMPYYEATISEDYYVESKPDVNSTDKEL LVAGTRVRVYEKVKGWARIGAPQSN QWVEDAYLIDATDM(SEQ ID NO: 59) S. agalactiae LambdaSa2 LambdaSa2 Glycosidase andMEINTEIAIAWMSARQGKVSYSMDY prophage endopeptidaseRDGPNSYDCSSSVYYALRSAGASSA GWAVNTEYMHDWLIKNGYELIAENVDWNAVRGDIAIWGMRGHSSGAGGHV VMFIDPENIIHCNWANNGITVNNYNQTAAASGWMYCYVYRLKSGASTQGK SLDTLVKETLAGNYGNGEARKAVLGNQYEAVMSVINGKTTTNQKTVDQLV QEVIAGKHGNGEARKKSLGSQYDAVQKRVTELLKKQPSEPFKAQEVNKPT ETKTSQTELTGQATATKEEGDLSFNGTILKKAVLDKILGNCKKHDILPSY ALTILHYEGLWGTSAVGKADNNWGGMTWTGQGNRPSGVTVTQGSARPSNE GGHYMHYASVDDFLTDWFYLLRAGGSYKVSGAKTFSEAIKGMFKVGGAVY DYAASGFDSYIVGASSRLKAIEAENGSLDKFDKATDIGDGSKDKIDITIE GIEVTINGITYELTKKPV (SEQ ID NO: 60) S. uberis(ATCC700407) Ply700 Amidase MTDSIQEMRKLQSIPVRYDMGDRYG prophageNDADRDGRIEMDCSSAVSKALGISM TNNTETLQQALPAIGYGKIHDAVDGTFDMQAYDVIIWAPRDGSSSLGAFG HVLIATSPTTAIHCNYGSDGITENDYNYIWEINGRPREIVFRKGVTQTQA TVTSQFERELDVNARLTVSDKPYYEATLSEDYYVEAGPRIDSQDKELIKA GTRVRVYEKLNGWSRINHPESAQWV EDSYLVDATEM(SEQ ID NO: 61) S. suis SMP LySMP Glycosidase and N/A endopeptidaseB. anthracis Bcp1 PlyB Muramidase N/A S. aureus Phi11 and Phi11 lysinAmidase and MQAKLTKNEFIEWLKTSEGKQFNVD Phi12 endopeptidaseLWYGFQCFDYANAGWKVLFGLLLKG LGAKDIPFANNFDGLATVYQNTPDFLAQPGDMVVFGSNYGAGYGHVAWVI EATLDYIIVYEQNWLGGGWTDGIEQPGWGWEKVTRRQHAYDFPMWFIRPN FKSETAPRSVQSPTQAPKKETAKPQPKAVELKIIKDVVKGYDLPKRGSNP KGIVIHNDAGSKGATAEAYRNGLVNAPLSRLEAGIAHSYVSGNTVWQALD ESQVGWHTANQIGNKYYYGIEVCQSMGADNATFLKNEQATFQECARLLKK WGLPANRNTIRLHNEFTSTSCPHRSSVLHTGFDPVTRGLLPEDKRLQLKD YFIKQIRAYMDGKIPVATVSNESSASSNTVKPVASAWKRNKYGTYYMEES ARFTNGNQPITVRKVGPFLSCPVGYQFQPGGYCDYTEVMLQDGHVWVGYT WEGQRYYLPIRTWNGSAPPNQILGD LWGEIS(SEQ ID NO: 62) S. aureus ΦH5 LysH5 Amidase andMQAKLTKKEFIEWLKTSEGKQYNAD endopeptidase GWYGFQCFDYANAGWKALFGLLLKGVGAKDIPFANNFDGLATVYQNTPDF LAQPGDMVVFGSNYGAGYGHVAWVIEATLDYIIVYEQNWLGGGWTDGVQQ PGSGWEKVTRRQHAYDFPMWFIRPNFKSETAPRSVQSPTQASKKETAKPQ PKAVELKIIKDVVKGYDLPKRGSNPNFIVIHNDAGSKGATAEAYRNGLVN APLSRLEAGIAHSYVSGNTVWQALDESQVGWHTANQIGNKYGYGIEVCQS MGADNATFLKNEQATFQECARLLKKWGLPANRNTIRLHNEFTSTSCPHRS SVLHTGFDPVTRGLLPEDKRLQLKDYFIKQIRAYMDGKIPVATVSNDSSA SSNTVKPVASAWKRNKYGTYYMEESARFTNGNQPITVRKVGPFLSCPVGY QFQPGGYCDYTEVMLQDGHVWVGYTWEGQRYYLPIRTWNGSAPPNQILGD LWGEIS (SEQ ID NO: 63) S. warneri ΦWMY LysWMYAmidase and MKTKAQAKSWINSKIGKGIDWDGMY endopeptidaseGYQCMDEAVDYIHHVTDGKVTMWGN AIDAPKNNFQGLCTVYTNTPEFRPAYGDVIVWSYGTFATYGHIAIVVNPD PYGDLQYITVLEQNWNGNGIYKTEFATIRTHDYTGVSHFIRPKFADEVKE TAKTVNKLSVQKKIVTPKNSVERIKNYVKTSGYINGEHYELYNRGHKPKG VVIHNTAGTASATQEGQRLTNMTFQQLANGVAHVYIDKNTIYETLPEDRI AWHVAQQYGNTEFYGIEVCGSRNTDKEQFLANEQVAFQEAARRLKSWGMR ANRNTVRLHHTFSSTECPDMSMLLHTGYSMKNGKPTQDITNKCADYFMKQ INAYIDGKQPTSTVVGSSSSNKLKAKNKDKSTGWNTNEYGTLWKKEHATF TCGVRQGIVTRTTGPFTSCPQAGVLYYGQSVNYDTVCKQDGYVWISWTTS DGYDVWMPIRTWDRSTDKVSEIWGT IS (SEQ ID NO: 64)Streptococci ΦNCTC PlyGBS Muramidase and MATYQEYKSRSNGNAYDIDGSFGAQ (GBS)11261 endopeptidase CWDGYADYCKYLGLPYANCTNTGYA RDIWEQRHENGILNYFDEVEVMQAGDVAIFMVVDGVTPYSHVAIFDSDAG GGYGWFLGQNQGGANGAYNIVKIPYSATYPTAFRPKVFKNAVTVTGNIGL NKGDYFIDVSAYQQADLTTTCQQAGTTKTIIKVSESIAWLSDRHQQQANT SDPIGYYHFGRFGGDSALAQREADLFLSNLPSKKVSYLVIDYEDSASADK QANTNAVIAFMDKIASAGYKPIYYSYKPFTLNNIDYQKIIAKYPNSIWIA GYPDYEVRTEPLWEFFPSMDGVRWWQFTSVGVAGGLDKNIVLLADDSSKM DIPKVDKPQELTFYQKLATNTKLDNSNVPYYEATLSTDYYVESKPNASSA DKEFIKAGTRVRVYEKVNGWSRINH PESAQWVEDSYLVNATDM(SEQ ID NO: 65) C. perfringens Φ3626 Ply3626 Amidase N/A C. difficileΦCD27 CD27 lysin Amidase N/A E. faecalis Φ1 PlyV12 Amidase N/AA. naeslundii ΦAv-1- Av-1 lysin Putative N/A amidase/muramidaseL. gasseri ΦgaY LysgaY Muramidase N/A S. aureus ΦSA4 LysSA4 Amidase andN/A endopeptidase S. haemolyticus ΦSH2 SH2 Amidase and N/A endopeptidaseB. thuringiensis ΦBtCS33 PlyBt33 Amidase N/A L. monocytogenes ΦP40PlyP40 Amidase N/A L. monocytogenes ΦFWLLm3 LysZ5 AmidaseMVKYTVENKIIAGLPKGKLKGANFV IAHETANSKSTIDNEVSYMTRNWQNAFVTHFVGGGGRVVQVANVNYVSWG AGQYANSYSYAQVELCRTSNATTFKKDYEVYCQLLVDLAKKAGIPITLDS GSKTSDKGIKSHKWVADKLGGTTHQDPYAYLSSWGISKAQFASDLAKVSG GGNTGTAPAKPSTPSTNLDKLGLVDYMNAKKMDSSYSNRAKLAKQYGIAN YSGTASQNTTLLSKIKGGAPKPSTPAPKPSTSTAKKIYFPPNKGNWSVYP TNKAPVKANAIGAINPTKFGGLTYTIQKDRGNGVYEIQTDQFGRVQVYGA PSTGAVIKK (SEQ ID NO: 66) B. cereus ΦBPS13LysBPS13 Amidase MAKREKYIFDVEAEVGKAAKSIKSL EAELSKLQKLNKEIDATGGDRTEKEMLATLKAAKEVNAEYQKMQRILKDL SKYSGKVSRKEFNDSKVINNAKTSVQGGKVTDSFGQMLKNMERQINSVNK QFDNHRKAMVDRGQQYTPHLKTNRKDSQGNSNPSMMGRNKSTTQDMEKAV DKFLNGQNEATTGLNQALYQLKEISKLNRRSESLSRRASASGYMSFQQYS NFTGDRRTVQQTYGGLKTANRERVLELSGQATGISKELDRLNSKKGLTAR EGEERKKLMRQLEGIDAELTARKKLNSSLDETTSNMEKFNQSLVDAQVSV KPERGTMRGMMYERAPAIALAIGGAITATIGKLYSEGGNHSKAMRPDEMY VGQQTGAVGANWRPNRTATMRSGLGNHLGFTGQEMMEFQSNYLSANGYHG AEDMKAATTGQATFARATGLGSDEVKDFFNTAYRSGGIDGNQTKQFQNAF LGAMKQSGAVGREKDQLKALNGILSSMSQNRTVSNQDMMRTVGLQSAISS SGVASLQGTKGGALMEQLDNGIREGFNDPQMRVLFGQGTKYQGMGGRAAL RKQMEKGISDPDNLNTLIDASKASAGQDPAEQAEVLATLASKMGVNMSSD QARGLIDLQPSGKLTKENIDKVMKEGLKEGSIESAKRDKAYSESKASIDN SSEAATAKQATELNDMGSKLRQANAALGGLPAPLYTAIAAVVAFTAAVAG SALMFKGASWLKGGMASKYGGKGGKGGKGGGTGGGGGAGGAAATGAGAAA GAGGVGAAAAGEVGAGVAAGGAAAGAAAGGSKLAGVGKGFMKGAGKLMLP LGILMGASEIMQAPEEAKGSAIGSAVGGIGGGIAGGAATGAIAGSFLGPI GTAVGGIAGGIAGGFAGSSLGETIGGWFDSGPKEDASAADKAKADASAAA LAAAAGTSGAVGSSALQSQMAQGITGAPNMSQVGSMASALGISSGAMASA LGISSGQENQIQTMTDKENTNTKKANEAKKGDNLSYERENISMYERVLTR AEQILAQARAQNGIMGVGGGGTAGAGGGINGFTGGGKLQFLAAGQKWSSS NLQQHDLGFTDQNLTAEDLDKWIDSKAPQGSMMRGMGATFLKAGQEYGLD PRYLIAHAAEESGWGTSKIARDKGNFFGIGAFDDSPYSSAYEFKDGTGSA AERGIMGGAKWISEKYYGKGNTTLDKMKAAGYATNASWAPNIASIMAGAP TGSGSGNVTATINVNVKGDEKVSDKLKNSSDMKKAGKDIGSLLGFYSREM TIA (SEQ ID NO: 67) S. aureus ΦGH15 LysGH15Amidase and MAKTQAEINKRLDAYAKGTVDSPYR endopeptidaseIKKATSYDPSFGVMEAGAIDADGYY HAQCQDLITDYVLWLTDNKVRTWGNAKDQIKQSYGTGFKIHENKPSTVPK KGWIAVFTSGSYQQWGHIGIVYDGGNTSTFTILEQNWNGYANKKPTKRVD NYYGLTHFIEIPVKAGTTVKKETAKKSASKTPAPKKKATLKVSKNHINYT MDKRGKKPEGMVIHNDAGRSSGQQYENSLANAGYARYANGIAHYYGSEGY VWEAIDAKNQIAWHTGDGTGANSGNFRFAGIEVCQSMSASDAQFLKNEQA VFQFTAEKFKEWGLTPNRKTVRLHMEFVPTACPHRSMVLHTGFNPVTQGR PSQAIMNKLKDYFIKQIKNYMDKGTSSSTVVKDGKTSSASTPATRPVTGS WKKNQYGTWYKPENATFVNGNQPIVTRIGSPFLNAPVGGNLPAGATIVYD EVCIQAGHIWIGYNAYNGDRVYCPV RTCQGVPPNHIPGVAWGVFK(SEQ ID NO: 68) S. aureus ΦvB SauS- HydH5 Endopeptidase and N/A PLA88glycosidase E. faecalis ΦF168/08 Lys168 Endopeptidase N/A E. faecalisΦF170/08 Lys170 Amidase N/A S. aureus ΦP-27/HP P-27/HP Nonspecified N/AC. perfringens ΦSM101 Psm Muramidase N/A C. sporogenes Φ8074-B1 CS74LAmidase MKIGIDMGHTLSGADYGVVGLRPES VLTREVGTKVIYKLQKLGHVVVNCTVDKASSVSESLYTRYYRANQANVDL FISIHFNATPGGTGTEVYTYAGRQLGEATRIRQEFKSLGLRDRGTKDGSG LAVIRNTKAKAMLVECCFCDNPNDMKLYNSESFSNAIVKGITGKLPNGES GNNNQGGNKVKAVVIYNEGADRRGAEYLADYLNCPTISNSRTFDYSCVEH VYAVGGKKEQYTKYLKTLLSGANRY DTMQQILNFINGGK(SEQ ID NO: 69) S. typhimurium ΦSPN1S SPN1S GlycosidaseMDINQFRRASGINEQLAARWFPHIT TAMNEFGITKPDDQAMFIAQVGHESGGFTRLQENFNYSVNGLSGFIRAGR ITPDQANALGRKTYEKSLPLERQRAIANLVYSKRMGNNGPGDGWNYRGRG LIQITGLNNYRDCGNGLKVDLVAQPELLAQDEYAARSAAWFFSSKGCMKY TGDLVRVTQIINGGQNGIDDRRTRY AAARKVLAL(SEQ ID NO: 70) C. michiganensis ΦCMP1 CMP1 Peptidase N/AC. michiganensis ΦCN77 CN77 Peptidase MGYWGYPNGQIPNDKMALYRGCLLRADAAAQAYALQDAYTRATGKPLVIL EGYRDLTRQKYLRNLYLSGRGNIAAVPGLSNHGWGLACDFAAPLNSSGSE EHRWMRQNAPLFGFDWARGKADNEPWHWEYGNVPVSRWASLDVTPIDRND MADITEGQMQRIAVILLDTEIQTPLGPRLVKHALGDALLLGQANANSIAE VPDKTWDVLVDHPLAKNEDGTPLKVRLGDVAKYEPLEHQNTRDAIAKLGT LQFTDKQLATIGAGVKPIDEASLVK KIVDGVRALFGRAAA(SEQ ID NO: 71) A. baumannii ΦAB2 LysAB2 GlycosidaseMILTKDGFSIIRNELFGGKLDQTQV DAINFIVAKATESGLTYPEAAYLLATIYHETGLPSGYRTMQPIKEAGSDS YLRSKKYYPYIGYGYVQLTWKENYERIGKLIGVDLIKNPEKALEPLIAIQ IAIKGMLNGWFTGVGFRRKRPVSKYNKQQYVAARNIINGKDKAELIAKYA IIFERALRSL (SEQ ID NO: 72) B. cereus ΦB4 LysB4Endopeptidase MAMALQTLIDKANRKLNVSGMRKDV ADRTRAVITQMHAQGIYICVAQGFRSFAEQNALYAQGRTKPGSIVTNARG GQSNHNYGVAVDLCLYTQDGSDVIWTVEGNFRKVIAAMKAQGFKWGGDWV SFKDYPHFELYDVVGGQKPPADNGGAVDNGGGSGSTGGSGGGSTGGGSTG GGYDSSWFTKETGTFVTNTSIKLRTAPFTSADVIATLPAGSPVNYNGFGI EYDGYVWIRQPRSNGYGYLATGESK GGKRQNYWGTFK(SEQ ID NO: 73) P. aeruginosa ΦKMV KMV45 Nonspecified N/AC. tyrobutyricum ΦCTP1 Ctp1I Glycosidase MKKIADISNLNGNVDVKLLFNLGYIGIIAKASEGGTFVDKYYKQNYTNTK AQGKITGAYHFANFSTIAKAQQEANFFLNCIAGTTPDFVVLDLEQQCTGD ITDACLAFLNIVAKKFKCVVYCNSSFIKEHLNSKICAYPLWIANYGVATP AFTLWTKYAMWQFTEKGQVSGISGYIDFSYITDEFIKYIKGEDEVENLVV YNDGADQRAAEYLADRLACPTINNARKFDYSNVKNVYAVGGNKEQYTSYL TTLIAGSTRYTTMQAVLDYIKNLK (SEQ ID NO: 74)P. aeruginosa ΦEL EL188 Transglycosylase N/A P. aeruginosa ΦKZ KZ144Transglycosylase N/A S. aureus Ply187 Cell WallMALPKTGKPTAKQVVDWAINLIGSG Hydrolase VDVDGYYGRQCWDLPNYIFNRYWNFKTPGNARDMAWYRYPEGFKVFRNTS DFVPKPGDIAVWTGGNYNWNTWGHTGIVVGPSTKSYFYSVDQNWNNSNSY VGSPAAKIKHSYFGVTHFVRPAYKAEPKPTPPAQNNPAPKDPEPSKKPES NKPIYKVVTKILFTTAHIEHVKANRFVHYITKSDNHNNKPNKIVIKNTNT ALSTIDVYRYRDELDKDEIPHFFVDRLNVWACRPIEDSINGYHDSVVLSI TETRTALSDNFKMNEIECLSLAESILKANNKKMSASNIIVDNKAWRTFKL HTGKDSLKSSSFTSKDYQKAVNELIKLFNDKDKLLNNKPKDVVERIRIRT IVKENTKFVPSELKPRNNIRDKQDSKIDRVINNYTLKQALNIQYKLNPKP QTSNGVSWYNASVNQIKSAMDTTKIFNNNVQVYQFLKLNQYQGIPVDKLN KLLVGKGTLANQGHAFADGCKKYNINEIYLIAHRFLESANGTSFFASGKT GVYNYFGIGAFDNNPNNAMAFARSHGWTSPTKAIIGGAEFVGKGYFNVGQ NTLYRMRWNPQKPGTHQYATDISWAKVQAQMISAMYKEIGLTGDYFIYDQ YKK (SEQ ID NO: 75) P. uorescens ΦOBP OBPgp279Glycosidase N/A L. monocytogenes ΦP35 Ply35 AmidaseMARKFTKAELVAKAEKKVGGLKPDV KKAVLSAVKEAYDRYGIGIIVSQGYRSIAEQNGLYAQGRTKPGNIVTNAK GGQSNHNFGVAVDFAIDLIDDGKIDSWQPSATIVNMMKRRGFKWGGDWKS FTDLPHFEACDWYRGERKYKVDTSEWKKKENINIVIKDVGYFQDKPQFLN SKSVRQWKHGTKVKLTKHNSHWYTGVVKDGNKSVRGYIYHSMAKVTSKNS DGSVNATINAHAFCWDNKKLNGGDFINLKRGFKGITHPASDGFYPLYFAS RKKTFYIPRYMFDIKK (SEQ ID NO: 76) L. fermentumΦPYB5 Lyb5 Muramidase N/A S. pneumoniae ΦCP-7 Cpl-7 MuramidaseMVKKNDLFVDVASHQGYDISGILEE AGTTNTIIKVSESTSYLNPCLSAQVSQSNPIGFYHFAWFGGNEEEAEAEA RYFLDNVPTQVKYLVLDYEDHASASVQRNTTACLRFMQIIAEAGYTPIYY SYKPFTLDNVDYQQILAQFPNSLWIAGYGLNDGTANFEYFPSMDGIRWWQ YSSNPFDKNIVLLDDEKEDNINNENTLKSLTTVANEVIQGLWGNGQERYD SLANAGYDPQAVQDKVNEILNAREIADLTTVANEVIQGLWGNGQERYDSL ANAGYDPQAVQDKVNEILNAREIADLTTVANEVIQGLWGNGQERYDSLAN AGYDPQAVQDKVNELLS (SEQ ID NO: 77)P. chloroaphis201 Φ2-1 201y2-1gp229 Glycosidase N/A S. entericaΦPVP-SE1) PVP-SE1gp146 Glycosidase N/A Corynebacterium ΦBFK20 BKF20Amidase N/A E. faecalis ΦEFAP-1 EFAL-1 Amidase MKLKGILLSVVTTFGLLFGATNVQAYEVNNEFNLQPWEGSQQLAYPNKII LHETANPRATGRNEATYMKNNWFNAHTTAIVGDGGIVYKVAPEGNVSWGA GNANPYAPVQIELQHTNDPELFKANYKAYVDYTRDMGKKFGIPMTLDQGG SLWEKGVVSHQWVTDFVWGDHTDPYGYLAKMGISKAQLAHDLANGVSGNT ATPTPKPDKPKPTQPSKPSNKKRFNYRVDGLEYVNGMWQIYNEHLGKIDF NWTENGIPVEVVDKVNPATGQPTKDQVLKVGDYFNFQENSTGVVQEQTPY MGYTLSHVQLPNEFIWLFTDSKQAL MYQ (SEQ ID NO: 78)Lactobacilli lamdaSA2 LysA, LysA2, Nonspecified N/A and Lysga YS. aureus SAL-1 Nonspecified N/A

In some instances, the lysin is a functionally active variant of thelysins described herein. In some instances, the variant of the lysin hasat least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity, e.g., over a specified region or overthe entire sequence, to a sequence of a lysin described herein or anaturally occurring lysin.

In some instances, the lysin may be bioengineered to modulate itsbioactivity, e.g., increase or decrease or regulate, or to specify atarget microorganism. In some instances, the lysin is produced by thetranslational machinery (e.g. a ribosome, etc.) of a microbial cell. Insome instances, the lysin is chemically synthesized. In some instances,the lysin is derived from a polypeptide precursor. The polypeptideprecursor can undergo cleavage (for example, processing by a protease)to yield the polypeptide of the lysin itself. As such, in someinstances, the lysin is produced from a precursor polypeptide. In someinstances, the lysin includes a polypeptide that has undergonepost-translational modifications, for example, cleavage, or the additionof one or more functional groups.

The lysins described herein may be formulated in a composition for anyof the uses described herein. The compositions disclosed herein mayinclude any number or type (e.g., classes) of lysins, such as at leastabout any one of 1 lysin, 2, 3, 4, 5, 10, 15, 20, or more lysins. Asuitable concentration of each lysin in the composition depends onfactors such as efficacy, stability of the lysin, number of distinctlysin, the formulation, and methods of application of the composition.In some instances, each lysin in a liquid composition is from about 0.1ng/mL to about 100 mg/mL. In some instances, each lysin in a solidcomposition is from about 0.1 ng/g to about 100 mg/g. In some instances,wherein the composition includes at least two types of lysins, theconcentration of each type of lysin may be the same or different.

A modulating agent including a lysin as described herein can becontacted with the target host in an amount and for a time sufficientto: (a) reach a target level (e.g., a predetermined or threshold level)of lysin concentration inside a target host; (b) reach a target level(e.g., a predetermined or threshold level) of lysin concentration insidea target host gut; (c) reach a target level (e.g., a predetermined orthreshold level) of lysin concentration inside a target hostbacteriocyte; (d) modulate the level, or an activity, of one or moremicroorganism (e.g., endosymbiont) in the target host; or/and (e)modulate fitness of the target host.

(c) Antimicrobial Peptides

The modulating agent described herein may include an antimicrobialpeptide (AMP). Any AMP suitable for inhibiting a microorganism residentin the host may be used. AMPs are a diverse group of molecules, whichare divided into subgroups on the basis of their amino acid compositionand structure. The AMP may be derived or produced from any organism thatnaturally produces AMPs, including AMPs derived from plants (e.g.,copsin), insects (e.g., drosocin, scorpion peptide (e.g., Uy192, UyCT3,D3, D10, Uy17, Uy192), mastoparan, poneratoxin, cecropin, moricin,melittin), frogs (e.g., magainin, dermaseptin, aurein), and mammals(e.g., cathelicidins, defensins and protegrins). For example, the AMPmay be a scorpion peptide, such as Uy192 (5′-FLSTIWNGIKGLL-3′; SEQ IDNO: 227), UyCT3 (5′-LSAIWSGIKSLF-3; SEQ ID NO: 228), D3(5′-LWGKLWEGVKSLI-3′; SEQ ID NO: 229), and D10(5′-FPFLKLSLKIPKSAIKSAIKRL-3′; SEQ ID NO: 230), Uy17(5′-ILSAIWSGIKGLL-3′; SEQ ID NO: 231), or a combination thereof. In someinstances, the antimicrobial peptide may be one having at least 90%sequence identity (e.g., at least 90%, 92%, 94%, 96%, 98%, or 100%sequence identity) with one or more of the following: cecropin (SEQ IDNO: 82), melittin, copsin, drosomycin (SEQ ID NO: 93), dermcidin (SEQ IDNO: 81), andropin (SEQ ID NO: 83), moricin (SEQ ID NO: 84), ceratotoxin(SEQ ID NO: 85), abaecin (SEQ ID NO: 86), apidaecin (SEQ ID NO: 87),prophenin (SEQ ID NO: 88), indolicidin (SEQ ID NO: 89), protegrin (SEQID NO: 90), tachyplesin (SEQ ID NO: 91), or defensin (SEQ ID NO: 92) toa vector of a human pathogen. Non-limiting examples of AMPs are listedin Table 6.

TABLE 6 Examples of Antimicrobial Peptides Example Type CharacteristicAMP Sequence Anionic rich in glutamic and dermcidinSSLLEKGLDGAKKAVGGLGKLGKD peptides aspartic acid AVEDLESVGKGAVHDVKDVLDSVL(SEQ ID NO: 79) Linear cationic lack cysteine cecropin AKWKLFKKIEKVGQNIRDGIIKAGP α-helical AVAVVGQATQIAK peptides(SEQ ID NO: 80) andropin MKYFSVLVVLTLILAIVDQSDAFINLLDKVEDALHTGAQAGFKLIRPV ERGATPKKSEKPEK (SEQ ID NO: 81) moricinMNILKFFFVFIVAMSLVSCSTAAP AKIPIKAIKTVGKAVGKGLRAINI ASTANDVFNFLKPKKRKH(SEQ ID NO: 82) ceratotoxin MANLKAVFLICIVAFIALQCVVAEPAAEDSVVVKRSIGSALKKALPVA KKIGKIALPIAKAALPVAAGLVG (SEQ ID NO: 83)Cationic rich in proline, arginine, abaecin MKVVIFIFALLATICAAFAYVPLPpeptide phenylalanine, glycine, NVPQPGRRPFPTFPGQGPFNPKIK enriched fortryptophan WPQGY specific amino (SEQ ID NO: 84) acid apidaecinsKNFALAILVVTFVVAVFGNTNLDP PTRPTRLRREAKPEAEPGNNRPVYIPQPRPPHPRLRREAEPEAEPGNN RPVYIPQPRPPHPRLRREAELEAEPGNNRPVYISQPRPPHPRLRREAE PEAEPGNNRPVYIPQPRPPHPRLRREAELEAEPGNNRPVYISQPRPPH PRLRREAEPEAEPGNNRPVYIPQPRPPHPRLRREAEPEAEPGNNRPVY IPQPRPPHPRLRREAEPEAEPGNNRPVYIPQPRPPHPRLRREAKPEAK PGNNRPVYIPQPRPPHPRI (SEQ ID NO: 85) propheninMETQRASLCLGRWSLWLLLLALVV PSASAQALSYREAVLRAVDRLNEQSSEANLYRLLELDQPPKADEDPGT PKPVSFTVKETVCPRPTRRPPELCDFKENGRVKQCVGTVTLDQIKDPL DITCNEGVRRFPWWWPFLRRPRLRRQAFPPPNVPGPRFPPPNVPGPRF PPPNFPGPRFPPPNFPGPRFPPPNFPGPPFPPPIFPGPWFPPPPPFRP PPFGPPRFPGRR (SEQ ID NO: 86) indolicidinMQTQRASLSLGRWSLWLLLLGLVV PSASAQALSYREAVLRAVDQLNELSSEANLYRLLELDPPPKDNEDLGT RKPVSFTVKETVCPRTIQQPAEQCDFKEKGRVKQCVGTVTLDPSNDQF DLNCNELQSVILPWKWPWWPWRRG (SEQ ID NO: 87)Anionic and contain 1-3 disulfide bond protegrinMETQRASLCLGRWSLWLLLLALVV cationic PSASAQALSYREAVLRAVDRLNEQ peptides thatSSEANLYRLLELDQPPKADEDPGT contain PKPVSFTVKETVCPRPTRQPPELC cysteine andDFKENGRVKQCVGTVTLDQIKDPL form disulfide DITCNEVQGVRGGRLCYCRRRFCV bondsCVGRG (SEQ ID NO: 88) tachyplesins KWCFRVCYRGICYRRCR (SEQ ID NO: 89)defensin MKCATIVCTIAVVLAATLLNGSVQ AAPQEEAALSGGANLNTLLDELPEETHHAALENYRAKRATCDLASGFG VGSSLCAAHCIARRYRGGYCNSKA VCVCRN (SEQ ID NO: 90)drosomycin MMQIKYLFALFAVLMLVVLGANEA DADCLSGRYKGPCAVWDNETCRRVCKEEGRSSGHCSPSLKCWCEGC (SEQ ID NO: 91)

The AMP may be active against any number of target microorganisms. Insome instances, the AMP may have antibacterial and/or antifungalactivities. In some instances, the AMP may have a narrow-spectrumbioactivity or a broad-spectrum bioactivity. For example, some AMPstarget and kill only a few species of bacteria or fungi, while othersare active against both gram-negative and gram-positive bacteria as wellas fungi.

Further, the AMP may function through a number of known mechanisms ofaction. For example, the cytoplasmic membrane is a frequent target ofAMPs, but AMPs may also interfere with DNA and protein synthesis,protein folding, and cell wall synthesis. In some instances, AMPs withnet cationic charge and amphipathic nature disrupt bacterial membranesleading to cell lysis. In some instances, AMPs may enter cells andinteract with intracellular target to interfere with DNA, RNA, protein,or cell wall synthesis. In addition to killing microorganisms, AMPs havedemonstrated a number of immunomodulatory functions that are involved inthe clearance of infection, including the ability to alter host geneexpression, act as chemokines and/or induce chemokine production,inhibit lipopolysaccharide induced pro-inflammatory cytokine production,promote wound healing, and modulating the responses of dendritic cellsand cells of the adaptive immune response.

In some instances, the AMP is a functionally active variant of the AMPsdescribed herein. In some instances, the variant of the AMP has at least70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity, e.g., over a specified region or over the entiresequence, to a sequence of an AMP described herein or a naturallyderived AMP.

In some instances, the AMP may be bioengineered to modulate itsbioactivity, e.g., increase or decrease or regulate, or to specify atarget microorganism. In some instances, the AMP is produced by thetranslational machinery (e.g. a ribosome, etc.) of a cell. In someinstances, the AMP is chemically synthesized. In some instances, the AMPis derived from a polypeptide precursor. The polypeptide precursor canundergo cleavage (for example, processing by a protease) to yield thepolypeptide of the AMP itself. As such, in some instances, the AMP isproduced from a precursor polypeptide. In some instances, the AMPincludes a polypeptide that has undergone post-translationalmodifications, for example, cleavage, or the addition of one or morefunctional groups.

The AMPs described herein may be formulated in a composition for any ofthe uses described herein. The compositions disclosed herein may includeany number or type (e.g., classes) of AMPs, such as at least about anyone of 1 AMP, 2, 3, 4, 5, 10, 15, 20, or more AMPs. For example, thecompositions may include a cocktail of AMPs (e.g., a cocktail ofscorpion peptides, e.g., UyCT3, D3, D10, and Uy17). A suitableconcentration of each AMP in the composition depends on factors such asefficacy, stability of the AMP, number of distinct AMP in thecomposition, the formulation, and methods of application of thecomposition. In some instances, each AMP in a liquid composition is fromabout 0.1 ng/mL to about 100 mg/mL (about 0.1 ng/mL to about 1 ng/mL,about 1 ng/mL to about 10 ng/mL, about 10 ng/mL to about 100 ng/mL,about 100 ng/mL to about 1000 ng/mL, about 1 mg/mL to about 10 mg/mL,about 10 mg/mL to about 100 mg/mL). In some instances, each AMP in asolid composition is from about 0.1 ng/g to about 100 mg/g (about 0.1ng/g to about 1 ng/g, about 1 ng/g to about 10 ng/g, about 10 ng/g toabout 100 ng/g, about 100 ng/g to about 1000 ng/g, about 1 mg/g to about10 mg/g, about 10 mg/g to about 100 mg/g). In some instances, whereinthe composition includes at least two types of AMPs, the concentrationof each type of AMP may be the same or different.

A modulating agent including an AMP as described herein can be contactedwith the target host in an amount and for a time sufficient to: (a)reach a target level (e.g., a predetermined or threshold level) of AMPconcentration inside a target host; (b) reach a target level (e.g., apredetermined or threshold level) of AMP concentration inside a targethost gut; (c) reach a target level (e.g., a predetermined or thresholdlevel) of AMP concentration inside a target host bacteriocyte; (d)modulate the level, or an activity, of one or more microorganism (e.g.,endosymbiont) in the target host; or/and (e) modulate fitness of thetarget host.

As illustrated by Examples 20-22, AMPs, such as scorpion peptides, canbe used as modulating agents that target an endosymbiotic bacterium inan insect host to decrease the fitness of the host (e.g., as outlinedherein).

(d) Nodule C-rich Peptides

The modulating agent described herein may include a nodule C-richpeptide (NCR peptide). NCR peptides are produced in certain leguminousplants and play an important role in the mutualistic, nitrogen-fixingsymbiosis of the plants with bacteria from the Rhizobiaceae family(rhizobia), resulting in the formation of root nodules where plant cellscontain thousands of intracellular endosymbionts. NCR peptides possessanti-microbial properties that direct an irreversible, terminaldifferentiation process of bacteria, e.g., to permeabilize the bacterialmembrane, disrupt cell division, or inhibit protein synthesis. Forexample, in Medicago truncatula nodule cells infected with Sinorhizobiummeliloti, hundreds of NCR peptides are produced which directirreversible differentiation of the bacteria into large polyploidnitrogen-fixing bacteroids.). Non-limiting examples of NCR peptides arelisted in Table 7.

TABLE 7 Examples of NCR Peptides NAME Peptide sequenceProducer >gi|152218086|gb|ABS31477.1| MTKIVVFIYVVILLLTIFHVSAKKKRYIMedicago truncatula NCR 340 ECETHEDCSQVFMPPFVMRCVIHECKIF NGEHLRY(SEQ ID NO: 92) >gi|152218084|gb|ABS31476.1|MAKIMKFVYNMIPFLSIFIITLQVNVVV Medicago truncatula NCR 339CEIDADCPQICMPPYEVRCVNHRCGWVN TDDSLFLTQEFTRSKQYIIS(SEQ ID NO: 93) >gi|152218082|gb|ABS31475.1|MYKVVESIFIRYMHRKPNMTKFFKFVYT Medicago truncatula NCR 338MFILISLFLVVTNANAHNCTDISDCSSN HCSYEGVSLCMNGQCICIYE(SEQ ID NO: 94) >gi|152218080|gb|ABS31474.1|MVETLRLFYIMILFVSLCLVVVDGESKL Medicago truncatula NCR 337EQTCSEDFECYIKNPHVPFGHLRCFEGF CQQLNGPA(SEQ ID NO: 95) >gi|152218078|gb|ABS31473.1|MAKIVNFVYSMIVFLFLFLVATKAARGY Medicago truncatula NCR 336LCVTDSHCPPHMCPPGMEPRCVRRMCKC LPIGWRKYFVP(SEQ ID NO: 96) >gi|152218076|gb|ABS31472.1|MQIGKNMVETPKLDYVIIFFFLYFFFRQ Medicago truncatula NCR 335MIILRLNTTFRPLNFKMLRFWGQNRNIM KHRGQKVHFSLILSDCKTNKDCPKLRRA NVRCRKSYCVPI(SEQ ID NO: 97) >gi|152218074|gb|ABS31471.1|MLRLYLVSYFLLKRTLLVSYFSYFSTYI Medicago truncatula NCR 334IECKTDNDCPISQLKIYAWKCVKNGCHL FDVIPMMYE(SEQ ID NO: 98) >gi|152218072|gb|ABS31470.1|MAEILKFVYIVILFVSLLLIVVASEREC Medicago truncatula NCR 333VTDDDCEKLYPTNEYRMMCDSGYCMNLL NGKIIYLLCLKKKKFLIIISVLL(SEQ ID NO: 99) >gi|152218070|gb|ABS31469.1|MAEIIKFVYIMILCVSLLLIEVAGEECV Medicago truncatula NCR 332TDADCDKLYPDIRKPLMCSIGECYSLYK GKFSLSIISKTSFSLMVYNVVTLVICLR LAYISLLLKFL(SEQ ID NO: 100) >gi|152218068|gb|ABS31468.1|MAEILKDFYAMNLFIFLIILPAKIRGET Medicago truncatula NCR 331LSLTHPKCHHIMLPSLFITEVFQRVTDD GCPKPVNHLRVVKCIEHICEYGYNYRPDFASQIPESTKMPRKRE (SEQ ID NO: 101) >gi|152218066|gb|ABS31467.1|MVEILKNFYAMNLFIFLIILAVKIRGAH Medicago truncatula NCR 330FPCVTDDDCPKPVNKLRVIKCIDHICQY ARNLPDFASEISESTKMPCKGE(SEQ ID NO: 102) >gi|152218064|gb|ABS31466.1|MFHAQAENMAKVSNFVCIMILFLALFFI Medicago truncatula NCR 329TMNDAARFECREDSHCVTRIKCVLPRKP ECRNYACGCYDSNKYR(SEQ ID NO: 103) >gi|152218062|gb|ABS31465.1|MQMRQNMATILNFVFVIILFISLLLVVT Medicago truncatula NCR 328KGYREPFSSFTEGPTCKEDIDCPSISCV NPQVPKCIMFECHCKYIPTTLK(SEQ ID NO: 104) >gi|152218060|gb|ABS31464.1|MATILMYVYITILFISILTVLTEGLYEP Medicago truncatula NCR 327LYNFRRDPDCRRNIDCPSYLCVAPKVPR CIMFECHCKDIPSDH(SEQ ID NO: 105) >gi|152218058|gb|ABS31463.1|MTTSLKFVYVAILFLSLLLVVMGGIRRF Medicago truncatula NCR 326ECRQDSDCPSYFCEKLTVPKCFWSKCYC K(SEQ ID NO: 106) >gi|152218056|gb|ABS31462.1|MTTSLKFVYVAILFLSLLLVVMGGIRKK Medicago truncatula NCR 325ECRQDSDCPSYFCEKLTIAKCIHSTCLC K(SEQ ID NO: 107) >gi|152218054|gb|ABS31461.1|MQIGKNMVETPKLVYFIILFLSIFLCIT Medicago truncatula NCR 324VSNSSFSQIFNSACKTDKDCPKFGRVNV RCRKGNCVPI(SEQ ID NO: 108) >gi|152218046|gb|ABS31457.1|MTAILKKFINAVFLFIVLFLATTNVEDF Medicago truncatula NCR 320VGGSNDECVYPDVFQCINNICKCVSHHR T(SEQ ID NO: 109) >gi|152218044|gb|ABS31456.1|MQKRKNMAQIIFYVYALIILFSPFLAAR Medicago truncatula NCR 319LVFVNPEKPCVTDADCDRYRHESAIYSD MFCKDGYCFIDYHHDPYP(SEQ ID NO: 110) >gi|152218042|gb|ABS31455.1|MQMRKNMAQILFYVYALLILFTPFLVAR Medicago truncatula NCR 318IMVVNPNNPCVTDADCQRYRHKLATRMI CNQGFCLMDFTHDPYAPSLP(SEQ ID NO: 111) >gi|152218040|gb|ABS31454.1|MNHISKFVYALIIFLSIYLVVLDGLPIS Medicago truncatula NCR 317CKDHFECRRKINILRCIYRQEKPMCINS ICTCVKLL(SEQ ID NO: 112) >gi|152218038|gb|ABS31453.1|MQREKNMAKIFEFVYAMIIFILLFLVEK Medicago truncatula NCR 316NVVAYLKFECKTDDDCQKSLLKTYVWKC VKNECYFFAKK(SEQ ID NO: 113) >gi|152218036|gb|ABS31452.1|MAGIIKFVHVLIIFLSLFHVVKNDDGSF Medicago truncatula NCR 315CFKDSDCPDEMCPSPLKEMCYFLQCKCG VDTIA(SEQ ID NO: 114) >gi|152218034|gb|ABS31451.1|MANTHKLVSMILFIFLFLASNNVEGYVN Medicago truncatula NCR 314CETDADCPPSTRVKRFKCVKGECRWTRM SYA(SEQ ID NO: 115) >gi|152218032|gb|ABS31450.1|MQRRKKKAQVVMFVHDLIICIYLFIVIT Medicago truncatula NCR 313TRKTDIRCRFYYDCPRLEYHFCECIEDF CAYIRLN(SEQ ID NO: 116) >gi|152218030|gb|ABS31449.1|MAKVYMFVYALIIFVSPFLLATFRTRLP Medicago truncatula NCR 312CEKDDDCPEAFLPPVMKCVNRFCQYEIL E(SEQ ID NO: 117) >gi|152218028|gb|ABS31448.1|MIKQFSVCYIQMRRNMTTILKFPYIMVI Medicago truncatula NCR 310CLLLLHVAAYEDFEKEIFDCKKDGDCDH MCVTPGIPKCTGYVCFCFENL(SEQ ID NO: 118) >gi|152218026|gb|ABS31447.1|MQRSRNMTTIFKFAYIMIICVFLLNIAA Medicago truncatula NCR 309QEIENGIHPCKKNEDCNHMCVMPGLPWC HENNLCFCYENAYGNTR(SEQ ID NO: 119) >gi|152218024|gb|ABS31446.1|MTIIIKFVNVLIIFLSLFHVAKNDDNKL Medicago truncatula NCR 304LLSFIEEGFLCFKDSDCPYNMCPSPLKE MCYFIKCVCGVYGPIRERRLYQSHNPMI Q(SEQ ID NO: 120) >gi|152218022|gb|ABS31445.1|MRKNMTKILMIGYALMIFIFLSIAVSIT Medicago truncatula NCR 303GNLARASRKKPVDVIPCIYDHDCPRKLY FLERCVGRVCKYL(SEQ ID NO: 121) >gi|152218020|gb|ABS31444.1|MAHKLVYAITLFIFLFLIANNIEDDIFC Medicago truncatula NCR 301ITDNDCPPNTLVQRYRCINGKCNLSFVS YG(SEQ ID NO: 122) >gi|152218018|gb|ABS31443.1|MDETLKFVYILILFVSLCLVVADGVKNI Medicago truncatula NCR 300NRECTQTSDCYKKYPFIPWGKVRCVKGR CRLDM(SEQ ID NO: 123) >gi|152218016|gb|ABS31442.1|MAKIIKFVYVLAIFFSLFLVAKNVNGWT Medicago truncatula NCR 290CVEDSDCPANICQPPMQRMCFYGECACV RSKFCT(SEQ ID NO: 124) >gi|152218014|gb|ABS31441.1|MVKIIKFVYFMTLFLSMLLVTTKEDGSV Medicago truncatula NCR 289ECIANIDCPQIFMLPFVMRCINFRCQIV NSEDT(SEQ ID NO: 125) >gi|152218012|gb|ABS31440.1|MDEILKFVYTLIIFFSLFFAANNVDANI Medicago truncatula NCR 286MNCQSTFDCPRDMCSHIRDVICIFKKCK CAGGRYMPQVP(SEQ ID NO: 126) >gi|152218008|gb|ABS31438.1|MQRRKNMANNHMLIYAMIICLFPYLVVT Medicago truncatula NCR 278FKTAITCDCNEDCLNFFTPLDNLKCIDN VCEVFM(SEQ ID NO: 127) >gi|152218006|gb|ABS31437.1|MVNILKFIYVIIFFILMFFVLIDVDGHV Medicago truncatula NCR 266LVECIENRDCEKGMCKFPFIVRCLMDQC KCVRIHNLI(SEQ ID NO: 128) >gi|152218004|gb|ABS31436.1|MIIQFSIYYMQRRKLNMVEILKFSHALI Medicago truncatula NCR 265IFLFLSALVTNANIFFCSTDEDCTWNLC RQPWVQKCRLHMCSCEKN(SEQ ID NO: 129) >gi|152218002|gb|ABS31435.1|MDEVFKFVYVMIIFPFLILDVATNAEKI Medicago truncatula NCR 263RRCFNDAHCPPDMCTLGVIPKCSRFTIC IC(SEQ ID NO: 130) >gi|152218000|gb|ABS31434.1|MHRKPNMTKFFKFVYTMFILISLFLVVT Medicago truncatula NCR 244NANANNCTDTSDCSSNHCSYEGVSLCMN GQCICIYE(SEQ ID NO: 131) >gi|152217998|gb|ABS31433.1|MQMKKMATILKFVYLIILLIYPLLVVTE Medicago truncatula NCR 239ESHYMKFSICKDDTDCPTLFCVLPNVPK CIGSKCHCKLMVN(SEQ ID NO: 132) >gi|152217996|gb|ABS31432.1|MVETLRLFYIMILFVSLYLVVVDGVSKL Medicago truncatula NCR 237AQSCSEDFECYIKNPHAPFGQLRCFEGY CQRLDKPT(SEQ ID NO: 133) >gi|152217994|gb|ABS31431.1|MTTFLKVAYIMIICVFVLHLAAQVDSQK Medicago truncatula NCR 228RLHGCKEDRDCDNICSVHAVTKCIGNMC RCLANVK(SEQ ID NO: 134) >gi|152217992|gb|ABS31430.1|MRINRTPAIFKFVYTIIIYLFLLRVVAK Medicago truncatula NCR 224DLPFNICEKDEDCLEFCAHDKVAKCMLN ICFCF(SEQ ID NO: 135) >gi|152217990|gb|ABS31429.1|MAEILKILYVFIIFLSLILAVISQHPFT Medicago truncatula NCR 221PCETNADCKCRNHKRPDCLWHKCYCY (SEQ ID NO: 136) >gi|152217988|gb|ABS31428.1|MRKSMATILKFVYVIMLFIYSLFVIESF Medicago truncatula NCR 217GHRFLIYNNCKNDTECPNDCGPHEQAKC ILYACYCVE(SEQ ID NO: 137) >gi|152217986|gb|ABS31427.1|MNTILKFIFVVFLFLSIFLSAGNSKSYG Medicago truncatula NCR 209PCTTLQDCETHNWFEVCSCIDFECKCWS LL(SEQ ID NO: 138) >gi|152217984|gb|ABS31426.1|MAEIIKFVYIMILCVSLLLIAEASGKEC Medicago truncatula NCR 206VTDADCENLYPGNKKPMFCNNTGYCMSL YKEPSRYM(SEQ ID NO: 139) >gi|152217982|gb|ABS31425.1|MAKIIKFVYIMILCVSLLLIVEAGGKEC Medicago truncatula NCR 201VTDVDCEKIYPGNKKPLICSTGYCYSLY EEPPRYHK(SEQ ID NO: 140) >gi|152217980|gb|ABS31424.1|MAKVTKFGYIIIHFLSLFFLAMNVAGGR Medicago truncatula NCR 200ECHANSHCVGKITCVLPQKPECWNYACV CYDSNKYR(SEQ ID NO: 141) >gi|152217978|gb|ABS31423.1|MAKIFNYVYALIMFLSLFLMGTSGMKNG Medicago truncatula NCR 192CKHTGHCPRKMCGAKTTKCRNNKCQCV(SEQ ID NO: 142) >gi|152217976|gb|ABS31422.1|MTEILKFVCVMIIFISSFIVSKSLNGGG Medicago truncatula NCR 189KDKCFRDSDCPKHMCPSSLVAKCINRLC RCRRPELQVQLNP(SEQ ID NO: 143) >gi|152217974|gb|ABS31421.1|MAHIIMFVYALIYALIIFSSLFVRDGIP Medicago truncatula NCR 187CLSDDECPEMSHYSFKCNNKICEYDLGE MSDDDYYLEMSRE(SEQ ID NO: 144) >gi|152217972|gb|ABS31420.1|MYREKNMAKTLKFVYVIVLFLSLFLAAK Medicago truncatula NCR 181NIDGRVSYNSFIALPVCQTAADCPEGTR GRTYKCINNKCRYPKLLKPIQ(SEQ ID NO: 145) >gi|152217970|gb|ABS31419.1|MAHIFNYVYALLVFLSLFLMVTNGIHIG Medicago truncatula NCR 176CDKDRDCPKQMCHLNQTPKCLKNICKCV(SEQ ID NO: 146) >gi|152217968|gb|ABS31418.1|MAEILKCFYTMNLFIFLIILPAKIREHI Medicago truncatula NCR 175QCVIDDDCPKSLNKLLIIKCINHVCQYV GNLPDFASQIPKSTKMPYKGE(SEQ ID NO: 147) >gi|152217966|gb|ABS31417.1|MAYISRIFYVLIIFLSLFFVVINGVKSL Medicago truncatula NCR 173LLIKVRSFIPCQRSDDCPRNLCVDQIIP TCVWAKCKCKNYND(SEQ ID NO: 148) >gi|152217964|gb|ABS31416.1|MANVTKFVYIAIYFLSLFFIAKNDATAT Medicago truncatula NCR 172FCHDDSHCVTKIKCVLPRTPQCRNEACG CYHSNKFR(SEQ ID NO: 149) >gi|152217962|gb|ABS31415.1|MGEIMKFVYVMIIYLFMFNVATGSEFIF Medicago truncatula NCR 171TKKLTSCDSSKDCRSFLCYSPKFPVCKR GICECI(SEQ ID NO: 150) >gi|152217960|gb|ABS31414.1|MGEMFKFIYTFILFVHLFLVVIFEDIGH NCR 169 IKYCGIVDDCYKSKKPLFKIWKCVENVC VLWYK(SEQ ID NO: 151) >gi|152217958|gb|ABS31413.1|MARTLKFVYSMILFLSLFLVANGLKIFC Medicago truncatula NCR 165IDVADCPKDLYPLLYKCIYNKCIVFTRI PFPFDWI(SEQ ID NO: 152) >gi|152217956|gb|ABS31412.1|MANITKFVYIAILFLSLFFIGMNDAAIL Medicago truncatula NCR 159ECREDSHCVTKIKCVLPRKPECRNNACT CYKGGFSFHH(SEQ ID NO: 153) >gi|152217954|gb|ABS31411.1|MQRVKKMSETLKFVYVLILFISIFHVVI Medicago truncatula NCR 147VCDSIYFPVSRPCITDKDCPNMKHYKAK CRKGFCISSRVR(SEQ ID NO: 154) >gi|152217952|gb|ABS31410.1|MQIRKIMSGVLKFVYAIILFLFLFLVAR Medicago truncatula NCR 146EVGGLETIECETDGDCPRSMIKMWNKNY RHKCIDGKCEWIKKLP(SEQ ID NO: 155) >gi|152217950|gb|ABS31409.1|MFVYDLILFISLILVVTGINAEADTSCH Medicago truncatula NCR 145SFDDCPWVAHHYRECIEGLCAYRILY (SEQ ID NO: 156) >gi|152217948|gb|ABS31408.1MQRRKKSMAKMLKFFFAIILLLSLFLVA Medicago truncatula NCR 144TEVGGAYIECEVDDDCPKPMKNSHPDTY YKCVKHRCQWAWK(SEQ ID NO: 157) >gi|152217946|gb|ABS31407.1MFVYTLIIFLFPSHVITNKIAIYCVSDD Medicago truncatula NCR 140DCLKTFTPLDLKCVDNVCEFNLRCKGKC GERDEKFVFLKALKKMDQKLVLEEQGNAREVKIPKKLLFDRIQVPTPATKDQVEED DYDDDDEEEEEEEDDVDMWFHLPDVVCH(SEQ ID NO: 158) >gi|152217944|gb|ABS31406.1MAKFSMFVYALINFLSLFLVETAITNIR Medicago truncatula NCR 138CVSDDDCPKVIKPLVMKCIGNYCYFFMI YEGP(SEQ ID NO: 159) >gi|152217942|gb|ABS31405.1MAHKFVYAIILFIFLFLVAKNVKGYVVC Medicago truncatula NCR 136RTVDDCPPDTRDLRYRCLNGKCKSYRLS YG(SEQ ID NO: 160) >gi|152217940|gb|ABS31404.1MQRKKNMGQILIFVFALINFLSPILVEM Medicago truncatula NCR 129TTTTIPCTFIDDCPKMPLVVKCIDNFCN YFEIK(SEQ ID NO: 161) >gi|152217938|gb|ABS31403.1MAQTLMLVYALIIFTSLFLVVISRQTDI Medicago truncatula NCR 128PCKSDDACPRVSSHHIECVKGFCTYWKL D(SEQ ID NO: 162) >gi|152217936|gb|ABS31402.1MLRRKNTVQILMFVSALLIYIFLFLVIT Medicago truncatula NCR 127SSANIPCNSDSDCPWKIYYTYRCNDGFC VYKSIDPSTIPQYMTDLIFPR(SEQ ID NO: 163) >gi|152217934|gb|ABS31401.1MAVILKFVYIMIIFLFLLYVVNGTRCNR Medicago truncatula NCR 122DEDCPFICTGPQIPKCVSHICFCLSSGK EAY(SEQ ID NO: 164) >gi|152217932|gb|ABS31400.1MDAILKFIYAMFLFLFLFVTTRNVEALF Medicago truncatula NCR 121ECNRDFVCGNDDECVYPYAVQCIHRYCK CLKSRN(SEQ ID NO: 165) >gi|152217930|gb|ABS31399.1MQIGRKKMGETPKLVYVIILFLSIFLCT Medicago truncatula NCR 119NSSFSQMINFRGCKRDKDCPQFRGVNIR CRSGFCTPIDS(SEQ ID NO: 166) >gi|152217928|gb|ABS31398.1MQMRKNMAQILFYVYALLILFSPFLVAR Medicago truncatula NCR 118IMVVNPNNPCVTDADCQRYRHKLATRMV CNIGFCLMDFTHDPYAPSLP(SEQ ID NO: 167) >gi|152217926|gb|ABS31397.1MYVYYIQMGKNMAQRFMFIYALIIFLSQ Medicago truncatula NCR 111FFVVINTSDIPNNSNRNSPKEDVFCNSN DDCPTILYYVSKCVYNFCEYVV(SEQ ID NO: 168) >gi|152217924|gb|ABS31396.1|MAKIVNFVYSMIIFVSLFLVATKGGSKP Medicago truncatula NCR 103FLTRPYPCNTGSDCPQNMCPPGYKPGCE DGYCNHCYKRW(SEQ ID NO: 169) >gi|152217922|gb|ABS31395.1|MVRTLKFVYVIILILSLFLVAKGGGKKI Medicago truncatula NCR 101YCENAASCPRLMYPLVYKCLDNKCVKFM MKSRFV(SEQ ID NO: 170) >gi|152217920|gb|ABS31394.1|MARTLKFVYAVILFLSLFLVAKGDDVKI Medicago truncatula NCR 96KCVVAANCPDLMYPLVYKCLNGICVQFT LTFPFV(SEQ ID NO: 171) >gi|152217918|gb|ABS31393.1|MSNTLMFVITFIVLVTLFLGPKNVYAFQ Medicago truncatula NCR 94PCVTTADCMKTLKTDENIWYECINDFCI PFPIPKGRK(SEQ ID NO: 172) >gi|152217916|gb|ABS31392.1|MKRVVNMAKIVKYVYVIIIFLSLFLVAT Medicago truncatula NCR 93KIEGYYYKCFKDSDCVKLLCRIPLRPKC MYRHICKCKVVLTQNNYVLT(SEQ ID NO: 173) >gi|152217914|gb|ABS31391.1|MKRGKNMSKILKFIYATLVLYLFLVVTK Medicago truncatula NCR 90ASDDECKIDGDCPISWQKFHTYKCINQK CKWVLRFHEY(SEQ ID NO: 174) >gi|152217912|gb|ABS31390.1|MAKTLNFMFALILFISLFLVSKNVAIDI Medicago truncatula NCR 88FVCQTDADCPKSELSMYTWKCIDNECNL FKVMQQMV(SEQ ID NO: 175) >gi|152217910|gb|ABS31389.1|MANTHKLVSMILFIFLFLVANNVEGYVN Medicago truncatula NCR 86CETDADCPPSTRVKRFKCVKGECRWTRM SYA(SEQ ID NO: 176) >gi|152217908|gb|ABS31388.1|MAHFLMFVYALITCLSLFLVEMGHLSIH Medicago truncatula NCR 77CVSVDDCPKVEKPITMKCINNYCKYFVD HKL(SEQ ID NO: 177) >gi|152217906|gb|ABS31387.1|MNQIPMFGYTLIIFFSLFPVITNGDRIP Medicago truncatula NCR 76CVTNGDCPVMRLPLYMRCITYSCELFFD GPNLCAVERI(SEQ ID NO: 178) >gi|152217904|gb|ABS31386.1|MRKDMARISLFVYALIIFFSLFFVLTNG Medicago truncatula NCR 74ELEIRCVSDADCPLFPLPLHNRCIDDVC  HLFTS(SEQ ID NO: 179) >gi|152217902|gb|ABS31385.1|MAQILMFVYFLIIFLSLFLVESIKIFTE Medicago truncatula NCR 68HRCRTDADCPARELPEYLKCQGGM CRLLIKKD(SEQ ID NO: 180) >gi|152217900|gb|ABS31384.1|MARVISLFYALIIFLFLFLVATNGDLSP Medicago truncatula NCR 65CLRSGDCSKDECPSHLVPKCIGLTCYCI(SEQ ID NO: 181) >gi|152217898|gb|ABS31383.1|MQRRKNMAQILLFAYVFIISISLFLVVT Medicago truncatula NCR 62NGVKIPCVKDTDCPTLPCPLYSKCVDGF CKMLSI(SEQ ID NO: 182) >gi|152217896|gb|ABS31382.1|MNHISKFVYALIIFLSVYLVVLDGRPVS Medicago truncatula NCR 57CKDHYDCRRKVKIVGCIFPQEKPMCINS MCTCIREIVP(SEQ ID NO: 183) >gi|152217894|gb|ABS31381.1|MKSQNHAKFISFYKNDLFKIFQNNDSHF Medicago truncatula NCR 56KVFFALIIFLYTYLHVTNGVFVSCNSHI HCRVNNHKIGCNIPEQYLLCVNLFCLWL DY(SEQ ID NO: 184) >gi|152217892|gb|ABS31380.1|MTYISKVVYALIIFLSIYVGVNDCMLVT Medicago truncatula NCR 54CEDHFDCRQNVQQVGCSFREIPQCINSI CKCMKG(SEQ ID NO: 185) >gi|152217890|gb|ABS31379.1|MTHISKFVFALIIFLSIYVGVNDCKRIP Medicago truncatula NCR 53CKDNNDCNNNWQLLACRFEREVPRCINS ICKCMPM(SEQ ID NO: 186) >gi|152217888|gb|ABS31378.1|MVQTPKLVYVIVLLLSIFLGMTICNSSF Medicago truncatula NCR 43SHFFEGACKSDKDCPKLHRSNVRCRKGQ CVQI(SEQ ID NO: 187) >gi|152217886|gb|ABS31377.1|MTKILMLFYAMIVFHSIFLVASYTDECS Medicago truncatula NCR 28TDADCEYILCLFPIIKRCIHNHCKCVPM GSIEPMSTIPNGVHKFHIINN(SEQ ID NO: 188) >gi|152217884|gb|ABS31376.1|MAKTLNFVCAMILFISLFLVSKNVALYI Medicago truncatula NCR 26IECKTDADCPISKLNMYNWRCIKSSCHL YKVIQFMV(SEQ ID NO: 189) >gi|152217882|gb|ABS31375.1|MQKEKNMAKTFEFVYAMIIFILLFLVEN Medicago truncatula NCR 24NFAAYIIECQTDDDCPKSQLEMFAWKCV KNGCHLFGMYEDDDDP(SEQ ID NO: 190) >gi|152217880|gb|ABS31374.1|MAATRKFIYVLSHFLFLFLVTKITDARV Medicago truncatula NCR 21CKSDKDCKDIIIYRYILKCRNGECVKIK I(SEQ ID NO: 191) >gi|152217878|gb|ABS31373.1|MQRLDNMAKNVKFIYVIILLLFIFLVII Medicago truncatula NCR 20VCDSAFVPNSGPCTTDKDCKQVKGYIAR CRKGYCMQSVKRTWSSYSR(SEQ ID NO: 192) >gi|152217876|gb|ABS31372.1|MKFIYIMILFLSLFLVQFLTCKGLTVPC Medicago truncatula NCR 19ENPTTCPEDFCTPPMITRCINFICLCDG PEYAEPEYDGPEPEYDHKGDFLSVKPKIINENMMMRERHMMKEIEV (SEQ ID NO: 193) >gi|152217874|gb|ABS31371.1|MAQFLMFIYVLIIFLYLFYVEAAMFELT Medicago truncatula NCR 12KSTIRCVTDADCPNVVKPLKPKCVDGFC EYT(SEQ ID NO: 194) >gi|152217872|gb|ABS31370.1|MKMRIHMAQIIMFFYALIIFLSPFLVDR Medicago truncatula NCR 10RSFPSSFVSPKSYTSEIPCKATRDCPYE LYYETKCVDSLCTY (SEQ ID NO: 195)

Any NCR peptide known in the art is suitable for use in the methods orcompositions described herein. NCR peptide-producing plants include butare not limited to Pisum sativum (pea), Astragalus sinicus (IRLClegumes), Phaseolus vulgaris (bean), Vigna unguiculata (cowpea),Medicago truncatula (barrelclover), and Lotus japonicus. For example,over 600 potential NCR peptides are predicted from the M. truncatulagenome sequence and almost 150 different NCR peptides have been detectedin cells isolated from root nodules by mass spectrometry.

The NCR peptides described herein may be mature or immature NCRpeptides. Immature NCR peptides have a C-terminal signal peptide that isrequired for translocation into the endoplasmic reticulum and cleavedafter translocation. The N-terminus of a NCR peptide includes a signalpeptide, which may be cleavable, for targeting to a secretory pathway.NCR peptides are generally small peptides with disulfide bridges thatstabilize their structure. Mature NCR peptides have a length in therange of about 20 to about 60 amino acids, about 25 to about 55 aminoacids, about 30 to about 50 amino acids, about 35 to about 45 aminoacids, or any range therebetween. NCR peptides may include a conservedsequence of cysteine residues with the rest of the peptide sequencehighly variable. NCR peptides generally have about four or eightcysteines.

NCR peptides may be anionic, neutral, or cationic. In some instances,synthetic cationic NCR peptides having a pI greater than about eightpossess antimicrobial activities. For example, NCR247 (pI=10.15)(RNGCIVDPRCPYQQCRRPLYCRRR; SEQ ID NO: 196) and NCR335 (pI=11.22)(MAQFLLFVYSLIIFLSLFFGEAAFERTETRMLTIPCTSDDNCPKVISPCHTKCFDGFCGWYIEGSYEGP;SEQ ID NO: 197) are both effective against gram-negative andgram-positive bacteria as well as fungi. In some instances, neutraland/or anionic NCR peptides, such as NCR001, do not possessantimicrobial activities at a pI greater than about 8.

In some instances, the NCR peptide is effective to kill bacteria. Insome instances, the NCR peptide is effective to kill S. meliloti,Xenorhabdus spp, Photorhabdus spp, Candidatus spp, Buchnera spp,Blattabacterium spp, Baumania spp, Wigglesworthia spp, Wolbachia spp,Rickettsia spp, Orientia spp, Sodalis spp, Burkholderia spp, Cupriavidusspp, Frankia spp, Snirhizobium spp, Streptococcus spp, Wolinella spp,Xylella spp, Erwinia spp, Agrobacterium spp, Bacillus spp, Paenibacillusspp, Streptomyces spp, Micrococcus spp, Corynebacterium spp, Acetobacterspp, Cyanobacteria spp, Salmonella spp, Rhodococcus spp, Pseudomonasspp, Lactobacillus spp, Enterococcus spp, Alcaligenes spp, Klebsiellaspp, Paenibacillus spp, Arthrobacter spp, Corynebacterium spp,Brevibacterium spp, Thermus spp, Pseudomonas spp, Clostridium spp, orEscherichia spp.

In some instances, the NCR peptide is a functionally active variant of aNCR peptide described herein. In some instances, the variant of the NCRpeptide has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a specified regionor over the entire sequence, to a sequence of a NCR peptide describedherein or naturally derived NCR peptide.

In some instances, the NCR peptide may be bioengineered to modulate itsbioactivity, e.g., increase or decrease or regulate, or to specify atarget microorganism. In some instances, the NCR peptide is produced bythe translational machinery (e.g. a ribosome, etc.) of a cell. In someinstances, the NCR peptide is chemically synthesized. In some instances,the NCR peptide is derived from a polypeptide precursor. The polypeptideprecursor can undergo cleavage (for example, processing by a protease)to yield the NCR peptide itself. As such, in some instances, the NCRpeptide is produced from a precursor polypeptide. In some instances, theNCR peptide includes a polypeptide that has undergone post-translationalmodifications, for example, cleavage, or the addition of one or morefunctional groups.

The NCR peptide described herein may be formulated in a composition forany of the uses described herein. The compositions disclosed herein mayinclude any number or type of NCR peptides, such as at least about anyone of 1 NCR peptide, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, or moreNCR peptides. A suitable concentration of each NCR peptide in thecomposition depends on factors such as efficacy, stability of the NCRpeptide, number of distinct NCR peptide, the formulation, and methods ofapplication of the composition. In some instances, each NCR peptide in aliquid composition is from about 0.1 ng/mL to about 100 mg/mL. In someinstances, each NCR peptide in a solid composition is from about 0.1ng/g to about 100 mg/g. In some instances, wherein the compositionincludes at least two types of NCR peptides, the concentration of eachtype of NCR peptide may be the same or different.

A modulating agent including a NCR peptide as described herein can becontacted with the target host in an amount and for a time sufficientto: (a) reach a target level (e.g., a predetermined or threshold level)of NCR peptide concentration inside a target host; (b) reach a targetlevel (e.g., a predetermined or threshold level) of NCR peptideconcentration inside a target host gut; (c) reach a target level (e.g.,a predetermined or threshold level) of NCR peptide concentration insidea target host bacteriocyte; (d) modulate the level, or an activity, ofone or more microorganism (e.g., endosymbiont) in the target host;or/and (e) modulate fitness of the target host.

(e) Bacteriocyte Regulatory Peptides

The modulating agent described herein may include a bacteriocyteregulatory peptide (BRP). BRPs are peptides expressed in thebacteriocytes of insects. These genes are expressed first at adevelopmental time point coincident with the incorporation of symbiontsand their bacteriocyte-specific expression is maintained throughout theinsect's life. In some instances, the BRP has a hydrophobic aminoterminal domain, which is predicted to be a signal peptide. In addition,some BRPs have a cysteine-rich domain. In some instances, thebacteriocyte regulatory peptide is a bacteriocyte-specific cysteine rich(BCR) protein. Bacteriocyte regulatory peptides have a length betweenabout 40 and 150 amino acids. In some instances, the bacteriocyteregulatory peptide has a length in the range of about 45 to about 145,about 50 to about 140, about 55 to about 135, about 60 to about 130,about 65 to about 125, about 70 to about 120, about 75 to about 115,about 80 to about 110, about 85 to about 105, or any range therebetween.Non-limiting examples of BRPs and their activities are listed in Table8.

TABLE 8 Examples of Bacteriocyte Regulatory Peptides NamePeptide Sequence Bacteriocyte-specific cysteine richMKLLHGFLIIMLTMHLSIQYAYGGPFLTKYLCDRVCH proteins BCR family, peptide BCR1KLCGDEFVCSCIQYKSLKGLWFPHCPTGKASVVLHNF LTSP (SEQ ID NO: 198)Bacteriocyte-specific cysteine richMKLLYGFLIIMLTIHLSVQYFESPFETKYNCDTHCNK proteins BCR family, peptide BCR2LCGKIDHCSCIQYHSMEGLWFPHCRTGSAAQMLHDFL SNP (SEQ ID NO: 199)Bacteriocyte-specific cysteine richMSVRKNVLPTMFVVLLIMSPVTPTSVFISAVCYSGCG proteins BCR family, peptide BCR3SLALVCFVSNGITNGLDYFKSSAPLSTSETSCGEAFD TCTDHCLANFKF (SEQ ID NO: 200)Bacteriocyte-specific cysteine richMRLLYGFLIIMLTIYLSVQDFDPTEFKGPFPTIEICS proteins BCR family, peptide BCR4KYCAVVCNYTSRPCYCVEAAKERDQWFPYCYD (SEQ ID NO: 201)Bacteriocyte-specific cysteine richMRLLYGFLIIMLTIHLSVQDIDPNTLRGPYPTKEICS proteins BCR family, peptide BCR5KYCEYNVVCGASLPCICVODAROLDHWFACCYDGGPE MLM (SEQ ID NO: 202)Secreted proteins SP family, peptideMKLFVVVVLVAVGIMFVFASDTAAAPTDYEDTNDMIS SP1LSSLVGDNSPYVRVSSADSGGSSKTSSKNPILGLLKS VIKLLTKIFGTYSDAAPAMPPIPPALRKNRGMLA(SEQ ID NO: 203) Secreted proteins SP family, peptideMVACKVILAVAVVFVAAVOGRPGGEPEWAAPIFAELK SP2SVSDNITNLVGLDNAGEYATAAKNNLNAFAESLKTEAAVFSKSFEGKASASDVFKESTKNFQAVVDTYIKNLPKDLTLKDFTEKSEQALKYMVEHGTEITKKAQGNTETEK EIKEFFKKQIENLIGQGKALQAKIAEAKKA(SEQ ID NO: 204) Secreted proteins SP family, peptideMKTSSSKVFASCVAIVCLASVANALPVQKSVAATTEN SP3PIVEKHGCRAHKNLVRQNVVDLKTYDSMLITNEVVQKQSNEVQSSEQSNEGQNSEQSNEGQNSEQSNEVQSSEHSNEGQNSKQSNEGQNSEQSNEVQSSEHSNEGQNSEQSNEVQSSEHSNEGQNSKQSNEGQNSKQSNEVQSSEHWNEGQNSKQSNEDQNSEQSNEGQNSKQSNEGQNSKQSNEDQNSEQSNEGQNSKQSNEVQSSEQSNEGQNSKQSNEGQSSEQSNEGQNSKQSNEVQSPEEHYDLPDPESSYESE ETKGSHESGDDSEHR (SEQ ID NO: 205)Secreted proteins SP family, peptideMKTIILGLCLFGALFWSTQSMPVGEVAPAVPAVPSEA SP4VPQKQVEAKPETNAASPVSDAKPESDSKPVDAEVKPTVSEVKAESEQKPSGEPKPESDAKPVVASESKPESDPKPAAVVESKPENDAVAPETNNDAKPENAAAPVSENKPATDAKAETELIAQAKPESKPASDLKAEPEAAKPNSEVPVALPLNPTETKATQQSVETNQVEQAAPAAAQADPAAAPAADPAPAPAAAPVAAEEAKLSESAPSTENKAAEEPSKPAEQQSAKPVEDAVPAASEISETKVSPAVPAVPEVPASPSAPAVADPVSAPEAEKNAEPAKAANSAEPAVQSEAKPAEDIQKSGAVVSAENPKPVEEQKPAEVAKPAEQSKSEAPAEAPKPTEQSAAEEPKKPESANDEKKEQHSVN KRDATKEKKPTDSIMKKQKQKKAN(SEQ ID NO: 206) Secreted proteins SP family, peptideMNGKIVLCFAVVFIGQAMSAATGTTPEVEDIKKVAEQ SP5aMSQTFMSVANHLVGITPNSADAQKSIEKIRTIMNKGFTDMETEANKMKDIVRKNADPKLVEKYDELEKELKKHLSTAKDMFEDKVVKPIGEKVELKKITENVIKTTKDMEA TMNKAIDGFKKQ (SEQ ID NO: 207)Secreted proteins SP family, peptideMHLFLALGLFIVCGMVDATFYNPRSQTFNQLMERRQR SP6SIPIPYSYGYHYNPIEPSINVLDSLSEGLDSRINTFKPIYQNVKMSTQDVNSVPRTQYQPKNSLYDSEYISAKDIPSLFPEEDSYDYKYLGSPLNKYLTRPSTQESGIAINLVAIKETSVFDYGFPTYKSPYSSDSVWNFGSKIPNTVFEDPQSVESDPNTFKVSSPTIKIVKLLPETPEQESIITTTKNYELNYKTTQETPTEAELYPITSEEFQTEDEWHPMVPKENTTKDESSFITTEEPLTEDKSNSITIEKTQTEDESNSIEFNSIRTEEKSNSITTEENQKEDDESMSTTSQETTTAFNLNDTFDTNRYSSSHESLMLRIRELMKNIADQQNKSQFRTVDNIPAKSQSNLSSDESTNQQFEPQL VNGADTYK (SEQ ID NO: 208)Colepotericin A, ColA peptide MTRTMLFLACVAALYVCISATAGKPEEFAKLSDEAPSNDQAMYESIQRYRRFVDGNRYNGGQQQQQQPKQWEVRPDLSRDQRGNTKAQVEINKKGDNHDINAGWGKNINGP DSHKDTWHVGGSVRW (SEQ ID NO: 209)RipA type I MKETTVVWAKLFLILIILAKPLGLKAVNECKRLGNNSCRSHGECCSGFCFIEPGWALGVCKRLGTPKKSDDSNNGKNIEKNNGVHERIDDVFERGVCSYYKGPSITANGDVFDENEMTAAHRTLPFNTMVKVEGMGTSVVVKINDRKTAADGKVMLLSRAAAESLNIDENTGPVQCQLKFVLDGSGCTPDYGDTCVLHHECCSQNCFREMFSDKGFCLPK (SEQ ID NO: 210)

In some instances, the BRP alters the growth and/or activity of one ormore bacteria resident in the bacteriocyte of the host. In someinstances, the BRP may be bioengineered to modulate its bioactivity(e.g., increase, decrease, or regulate) or to specify a targetmicroorganism. In some instances, the BRP is produced by thetranslational machinery (e.g. a ribosome, etc.) of a cell. In someinstances, the BRP is chemically synthesized. In some instances, the BRPis derived from a polypeptide precursor. The polypeptide precursor canundergo cleavage (for example, processing by a protease) to yield thepolypeptide of the BRP itself. As such, in some instances, the BRP isproduced from a precursor polypeptide. In some instances, the BRPincludes a polypeptide that has undergone post-translationalmodifications, for example, cleavage, or the addition of one or morefunctional groups.

Functionally active variants of the BRPs as described herein are alsouseful in the compositions and methods described herein. In someinstances, the variant of the BRP has at least 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g.,over a specified region or over the entire sequence, to a sequence of aBRP described herein or naturally derived BRP.

The BRP described herein may be formulated in a composition for any ofthe uses described herein. The compositions disclosed herein may includeany number or type (e.g., classes) of BRPs, such as at least about anyone of 1 BRP, 2, 3, 4, 5, 10, 15, 20, or more BRPs. A suitableconcentration of each BRP in the composition depends on factors such asefficacy, stability of the BRP, number of distinct BRP, the formulation,and methods of application of the composition. In some instances, eachBRP in a liquid composition is from about 0.1 ng/mL to about 100 mg/mL.In some instances, each BRP in a solid composition is from about 0.1ng/g to about 100 mg/g. In some instances, wherein the compositionincludes at least two types of BRPs, the concentration of each type ofBRP may be the same or different.

A modulating agent including a BRP as described herein can be contactedwith the target host in an amount and for a time sufficient to: (a)reach a target level (e.g., a predetermined or threshold level) of BRPconcentration inside a target host; (b) reach a target level (e.g., apredetermined or threshold level) of BRP concentration inside a targethost gut; (c) reach a target level (e.g., a predetermined or thresholdlevel) of BRP concentration inside a target host bacteriocyte; (d)modulate the level, or an activity, of one or more microorganism (e.g.,endosymbiont) in the target host; or/and (e) modulate fitness of thetarget host.

iii. Small Molecules

Numerous small molecules (e.g., an antibiotic or a metabolite) may beused in the compositions and methods described herein. In someinstances, an effective concentration of any small molecule describedherein may alter the level, activity, or metabolism of one or moremicroorganisms (as described herein) resident in a host, the alterationresulting in a decrease in the host's fitness.

A modulating agent comprising a small molecule as described herein canbe contacted with the target host in an amount and for a time sufficientto: (a) reach a target level (e.g., a predetermined or threshold level)of a small molecule concentration inside a target host; (b) reach atarget level (e.g., a predetermined or threshold level) of smallmolecule concentration inside a target host gut; (c) reach a targetlevel (e.g., a predetermined or threshold level) of a small moleculeconcentration inside a target host bacteriocyte; (d) modulate the level,or an activity, of one or more microorganism (e.g., endosymbiont) in thetarget host; or/and (e) modulate fitness of the target host.

The small molecules discussed hereinafter, namely antibiotics andsecondary metabolites, can be used to alter the level, activity, ormetabolism of target microorganisms as indicated in the sections fordecreasing the fitness of a host insect (e.g., vector of a humanpathogen), such as a mosquito, a mite, a louse, or a tick.

(a) Antibiotics

The modulating agent described herein may include an antibiotic. Anyantibiotic known in the art may be used. Antibiotics are commonlyclassified based on their mechanism of action, chemical structure, orspectrum of activity.

The antibiotic described herein may target any bacterial function orgrowth processes and may be either bacteriostatic (e.g., slow or preventbacterial growth) or bactericidal (e.g., kill bacteria). In someinstances, the antibiotic is a bactericidal antibiotic. In someinstances, the bactericidal antibiotic is one that targets the bacterialcell wall (e.g., penicillins and cephalosporins); one that targets thecell membrane (e.g., polymyxins); or one that inhibits essentialbacterial enzymes (e.g., rifamycins, lipiarmycins, quinolones, andsulfonamides). In some instances, the bactericidal antibiotic is anaminoglycoside. In some instances, the antibiotic is a bacteriostaticantibiotic. In some instances the bacteriostatic antibiotic targetsprotein synthesis (e.g., macrolides, lincosamides and tetracyclines).Additional classes of antibiotics that may be used herein include cycliclipopeptides (such as daptomycin), glycylcyclines (such as tigecycline),oxazolidinones (such as linezolid), or lipiarmycins (such asfidaxomicin). Examples of antibiotics include oxytetracycline,doxycycline, rifampicin, ciprofloxacin, ampicillin, and polymyxin B.Other non-limiting examples of antibiotics are found in Table 9.

TABLE 9 Examples of Antibiotics Antibiotics Action Penicillins,cephalosporins, Cell wall synthesis vancomycin Membrane active agent,disrupt Polymixin, gramicidin cell membrane Tetracyclines, macrolides,Inhibit protein synthesis chloramphenicol, clindamycin, spectinomycinSulfonamides Inhibit folate-dependent pathways Ciprofloxacin InhibitDNA-gyrase Isoniazid, rifampicin, Antimycobacterial agents pyrazinamide,ethambutol, (myambutol)l, streptomycin

The antibiotic described herein may have any level of target specificity(e.g., narrow- or broad-spectrum). In some instances, the antibiotic isa narrow-spectrum antibiotic, and thus targets specific types ofbacteria, such as gram-negative or gram-positive bacteria.Alternatively, the antibiotic may be a broad-spectrum antibiotic thattargets a wide range of bacteria.

The antibiotics described herein may be formulated in a composition forany of the uses described herein. The compositions disclosed herein mayinclude any number or type (e.g., classes) of antibiotics, such as atleast about any one of 1 antibiotic, 2, 3, 4, 5, 10, 15, 20, or moreantibiotics (e.g., a combination of rifampicin and doxycycline, or acombination of ampicillin and rifampicin). A suitable concentration ofeach antibiotic in the composition depends on factors such as efficacy,stability of the antibiotic, number of distinct antibiotics, theformulation, and methods of application of the composition. In someinstances, wherein the composition includes at least two types ofantibiotics, the concentration of each type of antibiotic may be thesame or different.

A modulating agent including an antibiotic as described herein can becontacted with the target host in an amount and for a time sufficientto: (a) reach a target level (e.g., a predetermined or threshold level)of antibiotic concentration inside a target host; (b) reach a targetlevel (e.g., a predetermined or threshold level) of antibioticconcentration inside a target host gut; (c) reach a target level (e.g.,a predetermined or threshold level) of antibiotic concentration inside atarget host bacteriocyte; (d) modulate the level, or an activity, of oneor more microorganism (e.g., endosymbiont) in the target host; or/and(e) modulate fitness of the target host.

As illustrated by Examples 1-4, 10, 14, 26, and 27, antibiotics (e.g.,doxycycline, oxytetracycline, azithromycin, ciprofloxacin, orrifampicin) can be used as modulating agents that target anendosymbiotic bacterium, such as a Wolbachia spp., in an insect host(e.g., an insect vector of an animal pathogen), such as a mosquito ormite or tick or biting louse, to decrease the fitness of the host (e.g.,as outlined herein). As illustrated by Example 3, antibiotics such asoxytetracycline can be used as modulating agents that target anendosymbiotic bacterium, such as a Rickettsia spp., in an insect host,such as ticks, to decrease the fitness of the host (e.g., as outlinedherein).

(b) Secondary Metabolites

In some instances, the modulating agent of the compositions and methodsdescribed herein includes a secondary metabolite. Secondary metabolitesare derived from organic molecules produced by an organism. Secondarymetabolites may act (i) as competitive agents used against bacteria,fungi, amoebae, plants, insects, and large animals; (ii) as metaltransporting agents; (iii) as agents of symbiosis between microbes andplants, insects, and higher animals; (iv) as sexual hormones; and (v) asdifferentiation effectors. Non-limiting examples of secondarymetabolites are found in Table 10.

TABLE 10 Examiles of Secondary Metabolites Phenyl- propanoids AlkaloidsTerpenoids Quinones Steroids Polyketides Anthocyanins AcridinesCarotenes Anthro- Cardiac Erythromycin quinones Coumarins BetalainesMonoterpenes Bezo- Glycosides Lovastatin and quinones other statinsFlavonoids Quinolozidines Sesquiterpenes Naphtho- Pregnen-oloneDiscoder-molide quinones Hydroxy- Furono- Diterpenes DerivativesAflatoxin B1 cinnamoyl quinones Derivatives Harring- TriterpenesAvermectins tonines Isoflavonoids Isoquino- Nystatin lines LignansIndoles Rifamycin Phenolenones Purines Proantho- Pyridines cyanidinsStilbenes Tropane Tanins Alkaloids

The secondary metabolite used herein may include a metabolite from anyknown group of secondary metabolites. For example, secondary metabolitescan be categorized into the following groups: alkaloids, terpenoids,flavonoids, glycosides, natural phenols (e.g., gossypol acetic acid),enals (e.g., trans-cinnamaldehyde), phenazines, biphenols anddibenzofurans, polyketides, fatty acid synthase peptides, nonribosomalpeptides, ribosomally synthesized and post-translationally modifiedpeptides, polyphenols polysaccharides (e.g., chitosan), and biopolymers.For an in-depth review of secondary metabolites see, for example,Vining, Annu. Rev. Microbiol. 44:395-427, 1990.

Secondary metabolites useful for compositions and methods describedherein include those that alter a natural function of an endosymbiont(e.g., primary or secondary endosymbiont), bacteriocyte, orextracellular symbiont. In some instances, one or more secondarymetabolites described herein is isolated from a high throughputscreening (HTS) for antimicrobial compounds. For example, a HTS screenidentified 49 antibacterial extracts that have specificity against grampositive and gram negative bacteria from over 39,000 crude extracts fromorganisms growing in diverse ecosystems of one specific region. In someinstances, the secondary metabolite is transported inside abacteriocyte.

In some instances, the small molecule is an inhibitor of vitaminsynthesis. In some instances, the vitamin synthesis inhibitor is avitamin precursor analog. In certain instances, the vitamin precursoranalog is pantothenol.

In some instances, the small molecule is an amino acid analog. Incertain instances, the amino acid analog is L-canvanine, D-arginine,D-valine, D-methionine, D-phenylalanine, D-histidine, D-tryptophan,D-threonine, D-leucine, L-NG-nitroarginine, or a combination thereof.

In some instances the small molecule is a natural antimicrobialcompound, such as propionic acid, levulinic acid, trans-cinnemaldehdye,nisin, or low molecular weight chitosan.

The secondary metabolite described herein may be formulated in acomposition for any of the uses described herein. The compositionsdisclosed herein may include any number or type (e.g., classes) ofsecondary metabolites, such as at least about any one of 1 secondarymetabolite, 2, 3, 4, 5, 10, 15, 20, or more secondary metabolites. Asuitable concentration of each secondary metabolite in the compositiondepends on factors such as efficacy, stability of the secondarymetabolite, number of distinct secondary metabolites, the formulation,and methods of application of the composition. In some instances,wherein the composition includes at least two types of secondarymetabolites, the concentration of each type of secondary metabolite maybe the same or different.

A modulating agent including a secondary metabolite as described hereincan be contacted with the target host in an amount and for a timesufficient to: (a) reach a target level (e.g., a predetermined orthreshold level) of secondary metabolite concentration inside a targethost; (b) reach a target level (e.g., a predetermined or thresholdlevel) of secondary metabolite concentration inside a target host gut;(c) reach a target level (e.g., a predetermined or threshold level) ofsecondary metabolite concentration inside a target host bacteriocyte;(d) modulate the level, or an activity, of one or more microorganism(e.g., endosymbiont) in the target host; or/and (e) modulate fitness ofthe target host.

As illustrated by Example 15, secondary metabolites (e.g., gossypol) canbe used as modulating agents that target an endosymbiotic bacterium inan insect host to decrease the fitness of the host (e.g., as outlinedherein). As further illustrated by Examples 11-13, 17-19, 23, and 24,small molecules, such as trans-cinnemaldehyde, levulinic acid, chitosan,vitamin analogs, or amino acid transport inhibitors, can also be used asmodulating agents that target an endosymbiotic bacterium in an insecthost to decrease the fitness of the host (e.g., as outlined herein).

iv. Bacteria as Modulating Agents

In some instances, the modulating agent described herein includes one ormore bacteria. Numerous bacteria are useful in the compositions andmethods described herein. In some instances, the agent is a bacterialspecies endogenously found in the host. In some instances, the bacterialmodulating agent is an endosymbiotic bacterial species. Non-limitingexamples of bacteria that may be used as modulating agents include allbacterial species described herein in Section II of the detaileddescription and those listed in Table 1. For example, the modulatingagent may be a bacterial species from any bacterial phyla present ininsect guts, including Gammaproteobacteria, Alphaproteobacteria,Betaproteobacteria, Bacteroidetes, Firmicutes (e.g., Lactobacillus andBacillus spp.), Clostridia, Actinomycetes, Spirochetes, Verrucomicrobia,and Actinobacteria.

In some instances, the modulating agent is a bacterium that disruptsmicrobial diversity or otherwise alters the microbiota of the host in amanner detrimental to the host. In one instance, bacteria may beprovided to disrupt the microbiota of mosquitos. For example, thebacterial modulating agent may compete with, displace, and/or reduce apopulation of symbiotic bacteria in a mosquito.

In another instance, bacteria may be provided to disrupt the microbiotaof mites. For example, the bacterial modulating agent may compete with,displace, and/or reduce a population of symbiotic bacteria in a mite.

In another instance, bacteria may be provided to disrupt the microbiotaof biting louse. For example, the bacterial modulating agent may competewith, displace, and/or reduce a population of symbiotic bacteria in abiting louse.

In another instance, bacteria may be provided to disrupt the microbiotaof ticks. For example, the bacterial modulating agent may compete with,displace, and/or reduce a population of symbiotic bacteria in a tick.

The bacterial modulating agents discussed herein can be used to alterthe level, activity, or metabolism of target microorganisms as indicatedin the sections for decreasing the fitness of a host insect (e.g., avector of a human pathogen), such as a mosquito a mite, a biting louse,or a tick.

v. Modifications to Modulating Agents

(a) Fusions

Any of the modulating agents described herein may be fused or linked toan additional moiety. In some instances, the modulating agent includes afusion of one or more additional moieties (e.g., 1 additional moiety, 2,3, 4, 5, 6, 7, 8, 9, 10, or more additional moieties). In someinstances, the additional moiety is any one of the modulating agentsdescribed herein (e.g., a peptide, polypeptide, small molecule, orantibiotic). Alternatively, the additional moiety may not act asmodulating agent itself but may instead serve a secondary function. Forexample, the additional moiety may to help the modulating agent access,bind, or become activated at a target site in the host (e.g., at a hostgut or a host bacteriocyte) or at a target microorganism resident in thehost (e.g., a vector of a human pathogen, e.g., a mosquito, a mite, abiting louse, or a tick).

In some instances, the additional moiety may help the modulating agentpenetrate a target host cell or target microorganism resident in thehost. For example, the additional moiety may include a cell penetratingpeptide. Cell penetrating peptides (CPPs) may be natural sequencesderived from proteins; chimeric peptides that are formed by the fusionof two natural sequences; or synthetic CPPs, which are syntheticallydesigned sequences based on structure-activity studies. In someinstances, CPPs have the capacity to ubiquitously cross cellularmembranes (e.g., prokaryotic and eukaryotic cellular membranes) withlimited toxicity. Further, CPPs may have the capacity to cross cellularmembranes via energy-dependent and/or independent mechanisms, withoutthe necessity of a chiral recognition by specific receptors. CPPs can bebound to any of the modulating agents described herein. For example, aCPP can be bound to an antimicrobial peptide (AMP), e.g., a scorpionpeptide, e.g., UY192 fused to a cell penetrating peptide (e.g.,YGRKKRRQRRRFLSTIWNGIKGLLFAM; SEQ ID NO: 232). Non-limiting examples ofCPPs are listed in Table 11.

TABLE 11 Examples of Cell Penetrating Peptides (CPPs) Peptide OriginSequence Protein-derived Penetratin Antennapedia RQIKIWFQNRRMKWKK(SEQ ID NO: 211) Tat peptide Tat GRKKRRQRRRPPQ (SEQ ID NO: 212) pVECCadherin LLIILRRRIRKQAHAHSK (SEQ ID NO: 213) Chimeric TransportanGalanine/Mastoparan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 214) MPGHIV-gp41/SV40 T-antigen GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 215)Pep-1 HIV-reverse KETWWETWWTEWSQPKKKRKV transcriptase/SV40(SEQ ID NO: 216) T-antigen Synthetic Polyarginines Based on Tat peptide(R)_(n); 6 < n < 12 MAP de novo KLALKLALKALKAALKLA (SEQ ID NO: 217) R₆W₃Based on penetratin RRWWRRWRR (SEQ ID NO: 218)

In other instances, the additional moiety helps the modulating agentbind a target microorganism (e.g., a fungi or bacterium) resident in thehost. The additional moiety may include one or more targeting domains.In some instances, the targeting domain may target the modulating agentto one or more microorganisms (e.g., bacterium or fungus) resident inthe gut of the host. In some instances, the targeting domain may targetthe modulating agent to a specific region of the host (e.g., host gut orbacteriocyte) to access microorganisms that are generally present insaid region of the host. For example, the targeting domain may targetthe modulating agent to the foregut, midgut, or hindgut of the host. Inother instances, the targeting domain may target the modulating agent toa bacteriocyte in the host and/or one or more specific bacteria residentin a host bacteriocyte. For example, the targeting domain may beGalanthus nivalis lectin or agglutinin (GNA) bound to a modulating agentdescribed herein, e.g., an AMP, e.g., a scorpion peptide, e.g., Uy192.

(b) Pre- or Pro-Domains

In some instances, the modulating agent may include a pre- or pro-aminoacid sequence. For example, the modulating agent may be an inactiveprotein or peptide that can be activated by cleavage orpost-translational modification of a pre- or pro-sequence. In someinstances, the modulating agent is engineered with an inactivating pre-or pro-sequence. For example, the pre- or pro-sequence may obscure anactivation site on the modulating agent, e.g., a receptor binding site,or may induce a conformational change in the modulating agent. Thus,upon cleavage of the pre- or pro-sequence, the modulating agent isactivated.

Alternatively, the modulating agent may include a pre- or pro-smallmolecule, e.g., an antibiotic. The modulating agent may be an inactivesmall molecule described herein that can be activated in a targetenvironment inside the host. For example, the small molecule may beactivated upon reaching a certain pH in the host gut.

(c) Linkers

In instances where the modulating agent is connected to an additionalmoiety, the modulating agent may further include a linker. For example,the linker may be a chemical bond, e.g., one or more covalent bonds ornon-covalent bonds. In some instances, the linker may be a peptidelinker (e.g., 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 20, 25, 30, 35, 40, ormore amino acids longer). The linker may be include any flexible, rigid,or cleavable linkers described herein.

A flexible peptide linker may include any of those commonly used in theart, including linkers having sequences having primarily Gly and Serresidues (“GS” linker). Flexible linkers may be useful for joiningdomains that require a certain degree of movement or interaction and mayinclude small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) aminoacids.

Alternatively, a peptide linker may be a rigid linker. Rigid linkers areuseful to keep a fixed distance between moieties and to maintain theirindependent functions. Rigid linkers may also be useful when a spatialseparation of the domains is critical to preserve the stability orbioactivity of one or more components in the fusion. Rigid linkers may,for example, have an alpha helix-structure or Pro-rich sequence,(XP)_(n), with X designating any amino acid, preferably Ala, Lys, orGlu.

In yet other instances, a peptide linker may be a cleavable linker. Insome instances, linkers may be cleaved under specific conditions, suchas the presence of reducing reagents or proteases. In vivo cleavablelinkers may utilize the reversible nature of a disulfide bond. Oneexample includes a thrombin-sensitive sequence (e.g., PRS) between twoCys residues. In vitro thrombin treatment of CPRSC results in thecleavage of the thrombin-sensitive sequence, while the reversibledisulfide linkage remains intact. Such linkers are known and described,e.g., in Chen et al., Adv. Drug Deliv. Rev. 65(10):1357-1369, 2013.Cleavage of linkers in fusions may also be carried out by proteases thatare expressed in vivo under conditions in specific cells or tissues ofthe host or microorganisms resident in the host. In some instances,cleavage of the linker may release a free functional, modulating agentupon reaching a target site or cell.

Fusions described herein may alternatively be linked by a linkingmolecule, including a hydrophobic linker, such as a negatively chargedsulfonate group; lipids, such as a poly (—CH2-) hydrocarbon chains, suchas polyethylene glycol (PEG) group, unsaturated variants thereof,hydroxylated variants thereof, amidated or otherwise N-containingvariants thereof, non-carbon linkers; carbohydrate linkers;phosphodiester linkers, or other molecule capable of covalently linkingtwo or more molecules, e.g., two modulating agents. Non-covalent linkersmay be used, such as hydrophobic lipid globules to which the modulatingagent is linked, for example, through a hydrophobic region of themodulating agent or a hydrophobic extension of the modulating agent,such as a series of residues rich in leucine, isoleucine, valine, orperhaps also alanine, phenylalanine, or even tyrosine, methionine,glycine, or other hydrophobic residue. The modulating agent may belinked using charge-based chemistry, such that a positively chargedmoiety of the modulating agent is linked to a negative charge of anothermodulating agent or an additional moiety.

IV. Formulations and Compositions

The compositions described herein may be formulated either in pure form(e.g., the composition contains only the modulating agent) or togetherwith one or more additional agents (such as excipient, delivery vehicle,carrier, diluent, stabilizer, etc.) to facilitate application ordelivery of the compositions. Examples of suitable excipients anddiluents include, but are not limited to, lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, saline solution, syrup,methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesiumstearate, and mineral oil.

In some instances, the composition includes a delivery vehicle orcarrier. In some instances, the delivery vehicle includes an excipient.Exemplary excipients include, but are not limited to, solid or liquidcarrier materials, solvents, stabilizers, slow-release excipients,colorings, and surface-active substances (surfactants). In someinstances, the delivery vehicle is a stabilizing vehicle. In someinstances, the stabilizing vehicle includes a stabilizing excipient.Exemplary stabilizing excipients include, but are not limited to,epoxidized vegetable oils, antifoaming agents, e.g. silicone oil,preservatives, viscosity regulators, binding agents and tackifiers. Insome instances, the stabilizing vehicle is a buffer suitable for themodulating agent. In some instances, the composition ismicroencapsulated in a polymer bead delivery vehicle. In some instances,the stabilizing vehicle protects the modulating agent against UV and/oracidic conditions. In some instances, the delivery vehicle contains a pHbuffer. In some instances, the composition is formulated to have a pH inthe range of about 4.5 to about 9.0, including for example pH ranges ofabout any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5to about 7.0.

Depending on the intended objectives and prevailing circumstances, thecomposition may be formulated into emulsifiable concentrates, suspensionconcentrates, directly sprayable or dilutable solutions, coatablepastes, diluted emulsions, spray powders, soluble powders, dispersiblepowders, wettable powders, dusts, granules, encapsulations in polymericsubstances, microcapsules, foams, aerosols, carbon dioxide gaspreparations, tablets, resin preparations, paper preparations, nonwovenfabric preparations, or knitted or woven fabric preparations. In someinstances, the composition is a liquid. In some instances, thecomposition is a solid. In some instances, the composition is anaerosol, such as in a pressurized aerosol can. In some instances, thecomposition is present in the waste (such as feces) of the pest. In someinstances, the composition is present in or on a live pest.

In some instances, the delivery vehicle is the food or water of thehost. In other instances, the delivery vehicle is a food source for thehost. In some instances, the delivery vehicle is a food bait for thehost. In some instances, the composition is a comestible agent consumedby the host. In some instances, the composition is delivered by the hostto a second host, and consumed by the second host. In some instances,the composition is consumed by the host or a second host, and thecomposition is released to the surrounding of the host or the secondhost via the waste (such as feces) of the host or the second host. Insome instances, the modulating agent is included in food bait intendedto be consumed by a host or carried back to its colony.

In some instances, the modulating agent may make up about 0.1% to about100% of the composition, such as any one of about 0.01% to about 100%,about 1% to about 99.9%, about 0.1% to about 10%, about 1% to about 25%,about 10% to about 50%, about 50% to about 99%, or about 0.1% to about90% of active ingredients (such as phage, lysin or bacteriocin). In someinstances, the composition includes at least any of 0.1%, 0.5%, 1%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more activeingredients (such as phage, lysin or bacteriocin). In some instances,the concentrated agents are preferred as commercial products, the finaluser normally uses diluted agents, which have a substantially lowerconcentration of active ingredient.

Any of the formulations described herein may be used in the form of abait, a coil, an electric mat, a smoking preparation, a fumigant, or asheet.

i. Liquid Formulations

The compositions provided herein may be in a liquid formulation. Liquidformulations are generally mixed with water, but in some instances maybe used with crop oil, diesel fuel, kerosene or other light oil as acarrier. The amount of active ingredient often ranges from about 0.5 toabout 80 percent by weight.

An emulsifiable concentrate formulation may contain a liquid activeingredient, one or more petroleum-based solvents, and an agent thatallows the formulation to be mixed with water to form an emulsion. Suchconcentrates may be used in agricultural, ornamental and turf, forestry,structural, food processing, livestock, and public health pestformulations. These may be adaptable to application equipment from smallportable sprayers to hydraulic sprayers, low-volume ground sprayers,mist blowers, and low-volume aircraft sprayers. Some active ingredientsare readily dissolve in a liquid carrier. When mixed with a carrier,they form a solution that does not settle out or separate, e.g., ahomogenous solution. Formulations of these types may include an activeingredient, a carrier, and one or more other ingredients. Solutions maybe used in any type of sprayer, indoors and outdoors.

In some instances, the composition may be formulated as an invertemulsion. An invert emulsion is a water-soluble active ingredientdispersed in an oil carrier. Invert emulsions require an emulsifier thatallows the active ingredient to be mixed with a large volume ofpetroleum-based carrier, usually fuel oil. Invert emulsions aid inreducing drift. With other formulations, some spray drift results whenwater droplets begin to evaporate before reaching target surfaces; as aresult the droplets become very small and lightweight. Because oilevaporates more slowly than water, invert emulsion droplets shrink lessand more active ingredient reaches the target. Oil further helps toreduce runoff and improve rain resistance. It further serves as asticker-spreader by improving surface coverage and absorption. Becausedroplets are relatively large and heavy, it is difficult to get thoroughcoverage on the undersides of foliage. Invert emulsions are mostcommonly used along rights-of-way where drift to susceptible non-targetareas can be a problem.

A flowable or liquid formulation combines many of the characteristics ofemulsifiable concentrates and wettable powders. Manufacturers use theseformulations when the active ingredient is a solid that does notdissolve in either water or oil. The active ingredient, impregnated on asubstance such as clay, is ground to a very fine powder. The powder isthen suspended in a small amount of liquid. The resulting liquid productis quite thick. Flowables and liquids share many of the features ofemulsifiable concentrates, and they have similar disadvantages. Theyrequire moderate agitation to keep them in suspension and leave visibleresidues, similar to those of wettable powders.

Flowables/liquids are easy to handle and apply. Because they areliquids, they are subject to spilling and splashing. They contain solidparticles, so they contribute to abrasive wear of nozzles and pumps.Flowable and liquid suspensions settle out in their containers. Becauseflowable and liquid formulations tend to settle, packaging in containersof five gallons or less makes remixing easier.

Aerosol formulations contain one or more active ingredients and asolvent. Most aerosols contain a low percentage of active ingredients.There are two types of aerosol formulations—the ready-to-use typecommonly available in pressurized sealed containers and those productsused in electrical or gasoline-powered aerosol generators that releasethe formulation as a smoke or fog.

Ready to use aerosol formulations are usually small, self-containedunits that release the formulation when the nozzle valve is triggered.The formulation is driven through a fine opening by an inert gas underpressure, creating fine droplets. These products are used ingreenhouses, in small areas inside buildings, or in localized outdoorareas. Commercial models, which hold five to 5 pounds of activeingredient, are usually refillable.

Smoke or fog aerosol formulations are not under pressure. They are usedin machines that break the liquid formulation into a fine mist or fog(aerosol) using a rapidly whirling disk or heated surface.

ii. Dry or Solid Formulations

Dry formulations can be divided into two types: ready-to-use andconcentrates that must be mixed with water to be applied as a spray.Most dust formulations are ready to use and contain a low percentage ofactive ingredients (less than about 10 percent by weight), plus a veryfine, dry inert carrier made from talc, chalk, clay, nut hulls, orvolcanic ash. The size of individual dust particles varies. A few dustformulations are concentrates and contain a high percentage of activeingredients. Mix these with dry inert carriers before applying. Dustsare always used dry and can easily drift to non-target sites.

iii. Granule or Pellet Formulations In some instances, the compositionis formulated as granules. Granular formulations are similar to dustformulations, except granular particles are larger and heavier. Thecoarse particles may be made from materials such as clay, corncobs, orwalnut shells. The active ingredient either coats the outside of thegranules or is absorbed into them. The amount of active ingredient maybe relatively low, usually ranging from about 0.5 to about 15 percent byweight. Granular formulations are most often used to apply to the soil,insects living in the soil, or absorption into plants through the roots.Granular formulations are sometimes applied by airplane or helicopter tominimize drift or to penetrate dense vegetation. Once applied, granulesmay release the active ingredient slowly. Some granules require soilmoisture to release the active ingredient. Granular formulations alsoare used to control larval mosquitoes and other aquatic pests. Granulesare used in agricultural, structural, ornamental, turf, aquatic,right-of-way, and public health (biting insect) pest-control operations.

In some instances, the composition is formulated as pellets. Most pelletformulations are very similar to granular formulations; the terms areused interchangeably. In a pellet formulation, however, all theparticles are the same weight and shape. The uniformity of the particlesallows use with precision application equipment.

iv. Powders

In some instances, the composition is formulated as a powder. In someinstances, the composition is formulated as a wettable powder. Wettablepowders are dry, finely ground formulations that look like dusts. Theyusually must be mixed with water for application as a spray. A fewproducts, however, may be applied either as a dust or as a wettablepowder—the choice is left to the applicator. Wettable powders have about1 to about 95 percent active ingredient by weight; in some cases morethan about 50 percent. The particles do not dissolve in water. Theysettle out quickly unless constantly agitated to keep them suspended.They can be used for most pest problems and in most types of sprayequipment where agitation is possible. Wettable powders have excellentresidual activity. Because of their physical properties, most of theformulation remains on the surface of treated porous materials such asconcrete, plaster, and untreated wood. In such cases, only the waterpenetrates the material.

In some instances, the composition is formulated as a soluble powder.Soluble powder formulations look like wettable powders. However, whenmixed with water, soluble powders dissolve readily and form a truesolution. After they are mixed thoroughly, no additional agitation isnecessary. The amount of active ingredient in soluble powders rangesfrom about 15 to about 95 percent by weight; in some cases more thanabout 50 percent. Soluble powders have all the advantages of wettablepowders and none of the disadvantages, except the inhalation hazardduring mixing.

In some instances, the composition is formulated as a water-dispersiblegranule. Water-dispersible granules, also known as dry flowables, arelike wettable powders, except instead of being dust-like, they areformulated as small, easily measured granules. Water-dispersiblegranules must be mixed with water to be applied. Once in water, thegranules break apart into fineparticles similar to wettable powders. Theformulation requires constant agitation to keep it suspended in water.The percentage of active ingredient is high, often as much as 90 percentby weight. Water-dispersible granules share many of the same advantagesand disadvantages of wettable powders, except they are more easilymeasured and mixed. Because of low dust, they cause less inhalationhazard to the applicator during handling

v. Bait

In some instances, the composition includes a bait. The bait can be inany suitable form, such as a solid, paste, pellet or powdered form. Thebait can also be carried away by the host back to a population of saidhost (e.g., a colony or hive). The bait can then act as a food sourcefor other members of the colony, thus providing an effective modulatingagent for a large number of hosts and potentially an entire host colony.

The baits can be provided in a suitable “housing” or “trap.” Suchhousings and traps are commercially available and existing traps can beadapted to include the compositions described herein. The housing ortrap can be box-shaped for example, and can be provided in pre-formedcondition or can be formed of foldable cardboard for example. Suitablematerials for a housing or trap include plastics and cardboard,particularly corrugated cardboard. The inside surfaces of the traps canbe lined with a sticky substance in order to restrict movement of thehost once inside the trap. The housing or trap can contain a suitabletrough inside which can hold the bait in place. A trap is distinguishedfrom a housing because the host cannot readily leave a trap followingentry, whereas a housing acts as a “feeding station” which provides thehost with a preferred environment in which they can feed and feel safefrom predators.

vi. Attractants

In some instances, the composition includes an attractant (e.g., achemoattractant). The attractant may attract an adult host or immaturehost (e.g., larva) to the vicinity of the composition. Attractantsinclude pheromones, a chemical that is secreted by an animal, especiallyan insect, which influences the behavior or development of others of thesame species. Other attractants include sugar and protein hydrolysatesyrups, yeasts, and rotting meat. Attractants also can be combined withan active ingredient and sprayed onto foliage or other items in thetreatment area.

Various attractants are known which influence host behavior as a host'ssearch for food, oviposition or mating sites, or mates. Attractantsuseful in the methods and compositions described herein include, forexample, eugenol, phenethyl propionate, ethyldimethylisobutyl-cyclopropane carboxylate, propylbenszodioxancarboxylate, cis-7,8-epoxy-2-methyloctadecane,trans-8,trans-0-dodecadienol, cis-9-tetradecenal (withcis-11-hexadecenal), trans-11-tetradecenal, cis-11-hexadecenal,(Z)-11,12-hexadecadienal, cis-7-dodecenyl acetate, cis-8-dodecenyulacetate, cis-9-dodecenyl acetate, cis-9-tetradecenyl acetate,cis-11-tetradecenyl acetate, trans-11-tetradecenyl acetate (withcis-11), cis-9,trans-11-tetradecadienyl acetate (with cis-9,trans-12),cis-9,trans-12-tetradecadienyl acetate, cis-7,cis-11-hexadecadienylacetate (with cis-7,trans-11), cis-3,cis-13-octadecadienyl acetate,trans-3,cis-13-octadecadienyl acetate, anethole and isoamyl salicylate.

Means other than chemoattractants may also be used to attract insects,including lights in various wavelengths or colors.

vii. Nanocapsules/Microencapsulation/Liposomes

In some instances, the composition is provided in a microencapsulatedformulation. Microencapsulated formulations are mixed with water andsprayed in the same manner as other sprayable formulations. Afterspraying, the plastic coating breaks down and slowly releases the activeingredient.

viii. Carriers

Any of the compositions described herein may be formulated to includethe modulating agent described herein and an inert carrier. Such carriercan be a solid carrier, a liquid carrier, a gel carrier, and/or agaseous carrier. In certain instances, the carrier can be a seedcoating. The seed coating is any non-naturally occurring formulationthat adheres, in whole or part, to the surface of the seed. Theformulation may further include an adjuvant or surfactant. Theformulation can also include one or more modulating agents to enlargethe action spectrum.

A solid carrier used for formulation includes finely-divided powder orgranules of clay (e.g. kaolin clay, diatomaceous earth, bentonite,Fubasami clay, acid clay, etc.), synthetic hydrated silicon oxide, talc,ceramics, other inorganic minerals (e.g., sericite, quartz, sulfur,activated carbon, calcium carbonate, hydrated silica, etc.), a substancewhich can be sublimated and is in the solid form at room temperature(e.g., 2,4,6-triisopropyl-1,3,5-trioxane, naphthalene,p-dichlorobenzene, camphor, adamantan, etc.); wool; silk; cotton; hemp;pulp; synthetic resins (e.g., polyethylene resins such as low-densitypolyethylene, straight low-density polyethylene and high-densitypolyethylene; ethylene-vinyl ester copolymers such as ethylene-vinylacetate copolymers; ethylene-methacrylic acid ester copolymers such asethylene-methyl methacrylate copolymers and ethylene-ethyl methacrylatecopolymers; ethylene-acrylic acid ester copolymers such asethylene-methyl acrylate copolymers and ethylene-ethyl acrylatecopolymers; ethylene-vinylcarboxylic acid copolymers such asethylene-acrylic acid copolymers; ethylene-tetracyclododecenecopolymers; polypropylene resins such as propylene homopolymers andpropylene-ethylene copolymers; poly-4-methylpentene-1, polybutene-1,polybutadiene, polystyrene; acrylonitrile-styrene resins; styreneelastomers such as acrylonitrile-butadiene-styrene resins,styrene-conjugated diene block copolymers, and styrene-conjugated dieneblock copolymer hydrides; fluororesins; acrylic resins such aspoly(methyl methacrylate); polyamide resins such as nylon 6 and nylon66; polyester resins such as polyethylene terephthalate, polyethylenenaphthalate, polybutylene terephthalate, andpolycyclohexylenedimethylene terephthalate; polycarbonates, polyacetals,polyacrylsulfones, polyarylates, hydroxybenzoic acid polyesters,polyetherimides, polyester carbonates, polyphenylene ether resins,polyvinyl chloride, polyvinylidene chloride, polyurethane, and porousresins such as foamed polyurethane, foamed polypropylene, or foamedethylene, etc.), glasses, metals, ceramics, fibers, cloths, knittedfabrics, sheets, papers, yarn, foam, porous substances, andmultifilaments.

A liquid carrier may include, for example, aromatic or aliphatichydrocarbons (e.g., xylene, toluene, alkylnaphthalene,phenylxylylethane, kerosine, gas oil, hexane, cyclohexane, etc.),halogenated hydrocarbons (e.g., chlorobenzene, dichloromethane,dichloroethane, trichloroethane, etc.), alcohols (e.g., methanol,ethanol, isopropyl alcohol, butanol, hexanol, benzyl alcohol, ethyleneglycol, etc.), ethers (e.g., diethyl ether, ethylene glycol dimethylether, diethylene glycol monomethyl ether, diethylene glycol monoethylether, propylene glycol monomethyl ether, tetrahydrofuran, dioxane,etc.), esters (e.g., ethyl acetate, butyl acetate, etc.), ketones (e.g.,acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone,etc.), nitriles (e.g., acetonitrile, isobutyronitrile, etc.), sulfoxides(e.g., dimethyl sulfoxide, etc.), amides (e.g., N,N-dimethylformamide,N,N-dimethylacetamide, cyclic imides (e.g. N-methylpyrrolidone)alkylidene carbonates (e.g., propylene carbonate, etc.), vegetable oil(e.g., soybean oil, cottonseed oil, etc.), vegetable essential oils(e.g., orange oil, hyssop oil, lemon oil, etc.), or water.

A gaseous carrier may include, for example, butane gas, flon gas,liquefied petroleum gas (LPG), dimethyl ether, and carbon dioxide gas.

ix. Adjuvants

In some instances, the composition provided herein may include anadjuvant. Adjuvants are chemicals that do not possess activity.Adjuvants are either pre-mixed in the formulation or added to the spraytank to improve mixing or application or to enhance performance. Theyare used extensively in products designed for foliar applications.Adjuvants can be used to customize the formulation to specific needs andcompensate for local conditions. Adjuvants may be designed to performspecific functions, including wetting, spreading, sticking, reducingevaporation, reducing volatilization, buffering, emulsifying,dispersing, reducing spray drift, and reducing foaming. No singleadjuvant can perform all these functions, but compatible adjuvants oftencan be combined to perform multiple functions simultaneously.

Among nonlimiting examples of adjuvants included in the formulation arebinders, dispersants and stabilizers, specifically, for example, casein,gelatin, polysaccharides (e.g., starch, gum arabic, cellulosederivatives, alginic acid, etc.), lignin derivatives, bentonite, sugars,synthetic water-soluble polymers (e.g., polyvinyl alcohol,polyvinylpyrrolidone, polyacrylic acid, etc.), PAP (acidic isopropylphosphate), BHT (2,6-di-t-butyl-4-methylphenol), BHA (a mixture of2-t-butyl-4-methoxyphenol and 3-t-butyl-4-methoxyphenol), vegetableoils, mineral oils, fatty acids and fatty acid esters.

x. Surfactants

In some instances, the composition provided herein includes asurfactant. Surfactants, also called wetting agents and spreaders,physically alter the surface tension of a spray droplet. For aformulation to perform its function properly, a spray droplet must beable to wet the foliage and spread out evenly over a leaf. Surfactantsenlarge the area of formulation coverage, thereby increasing the pest'sexposure to the chemical. Surfactants are particularly important whenapplying a formulation to waxy or hairy leaves. Without proper wettingand spreading, spray droplets often run off or fail to cover leafsurfaces adequately. Too much surfactant, however, can cause excessiverunoff and reduce efficacy.

Surfactants are classified by the way they ionize or split apart intoelectrically charged atoms or molecules called ions. A surfactant with anegative charge is anionic. One with a positive charge is cationic, andone with no electrical charge is nonionic. Formulation activity in thepresence of a nonionic surfactant can be quite different from activityin the presence of a cationic or anionic surfactant. Selecting the wrongsurfactant can reduce the efficacy of a pesticide product and injure thetarget plant. Anionic surfactants are most effective when used withcontact pesticides (pesticides that control the pest by direct contactrather than being absorbed systemically). Cationic surfactants shouldnever be used as stand-alone surfactants because they usually arephytotoxic.

Nonionic surfactants, often used with systemic pesticides, helppesticide sprays penetrate plant cuticles. Nonionic surfactants arecompatible with most pesticides, and most EPA-registered pesticides thatrequire a surfactant recommend a nonionic type. Adjuvants include, butare not limited to, stickers, extenders, plant penetrants, compatibilityagents, buffers or pH modifiers, drift control additives, defoamingagents, and thickeners.

Among nonlimiting examples of surfactants included in the compositionsdescribed herein are alkyl sulfate ester salts, alkyl sulfonates, alkylaryl sulfonates, alkyl aryl ethers and polyoxyethylenated productsthereof, polyethylene glycol ethers, polyvalent alcohol esters and sugaralcohol derivatives.

xi. Combinations

In formulations and in the use forms prepared from these formulations,the modulating agent may be in a mixture with other active compounds,such as pesticidal agents (e.g., insecticides, sterilants, acaricides,nematicides, molluscicides, or fungicides; see, e.g., pesticides listedin Table 12), attractants, growth-regulating substances, or herbicides.As used herein, the term “pesticidal agent” refers to any substance ormixture of substances intended for preventing, destroying, repelling, ormitigating any pest. A pesticide can be a chemical substance orbiological agent used against pests including insects, pathogens, weeds,and microbes that compete with humans for food, destroy property, spreaddisease, or are a nuisance. The term “pesticidal agent” may furtherencompass other bioactive molecules such as antibiotics, antiviralspesticides, antifungals, antihelminthics, nutrients, pollen, sucrose,and/or agents that stun or slow insect movement.

In instances where the modulating agent is applied to plants, a mixturewith other known compounds, such as herbicides, fertilizers, growthregulators, safeners, semiochemicals, or else with agents for improvingplant properties is also possible.

V. Delivery

A host described herein can be exposed to any of the compositionsdescribed herein in any suitable manner that permits delivering oradministering the composition to the insect. The modulating agent may bedelivered either alone or in combination with other active or inactivesubstances and may be applied by, for example, spraying, microinjection,through plants, pouring, dipping, in the form of concentrated liquids,gels, solutions, suspensions, sprays, powders, pellets, briquettes,bricks and the like, formulated to deliver an effective concentration ofthe modulating agent. Amounts and locations for application of thecompositions described herein are generally determined by the habits ofthe host, the lifecycle stage at which the microorganisms of the hostcan be targeted by the modulating agent, the site where the applicationis to be made, and the physical and functional characteristics of themodulating agent. The modulating agents described herein may beadministered to the insect by oral ingestion, but may also beadministered by means which permit penetration through the cuticle orpenetration of the insect respiratory system.

In some instances, the insect can be simply “soaked” or “sprayed” with asolution including the modulating agent. Alternatively, the modulatingagent can be linked to a food component (e.g., comestible) of the insectfor ease of delivery and/or in order to increase uptake of themodulating agent by the insect. Methods for oral introduction include,for example, directly mixing a modulating agent with the insects food,spraying the modulating agent in the insects habitat or field, as wellas engineered approaches in which a species that is used as food isengineered to express a modulating agent, then fed to the insect to beaffected. In some instances, for example, the modulating agentcomposition can be incorporated into, or overlaid on the top of, theinsects diet. For example, the modulating agent composition can besprayed onto a field of crops which an insect inhabits.

In some instances, the composition is sprayed directly onto a plante.g., crops, by e.g., backpack spraying, aerial spraying, cropspraying/dusting etc. In instances where the modulating agent isdelivered to a plant, the plant receiving the modulating agent may be atany stage of plant growth. For example, formulated modulating agents canbe applied as a seed-coating or root treatment in early stages of plantgrowth or as a total plant treatment at later stages of the crop cycle.In some instances, the modulating agent may be applied as a topicalagent to a plant, such that the host insect ingests or otherwise comesin contact with the plant upon interacting with the plant.

Further, the modulating agent may be applied (e.g., in the soil in whicha plant grows, or in the water that is used to water the plant) as asystemic agent that is absorbed and distributed through the tissues(e.g., stems or leafs) of a plant or animal host, such that an insectfeeding thereon will obtain an effective dose of the modulating agent.In some instances, plants or food organisms may be geneticallytransformed to express the modulating agent such that a host feedingupon the plant or food organism will ingest the modulating agent.

Delayed or continuous release can also be accomplished by coating themodulating agent or a composition containing the modulating agent(s)with a dissolvable or bioerodable coating layer, such as gelatin, whichcoating dissolves or erodes in the environment of use, to then make themodulating agent available, or by dispersing the agent in a dissolvableor erodable matrix. Such continuous release and/or dispensing meansdevices may be advantageously employed to consistently maintain aneffective concentration of one or more of the modulating agentsdescribed herein in a specific host habitat.

The modulating agent can also be incorporated into the medium in whichthe insect grows, lives, reproduces, feeds, or infests. For example, amodulating agent can be incorporated into a food container, feedingstation, protective wrapping, or a hive. For some applications themodulating agent may be bound to a solid support for application inpowder form or in a “trap” or “feeding station.” As an example, forapplications where the composition is to be used in a trap or as baitfor a particular host insect, the compositions may also be bound to asolid support or encapsulated in a time-release material. For example,the compositions described herein can be administered by delivering thecomposition to at least one habitat where a vector (e.g., a vector of ahuman pathogen, e.g., a mosquito, mite, biting louse, or tick) grows,lives, reproduces, feeds, or infests.

VI. Screening

Included herein are methods for screening for modulating agents that areeffective to alter the microbiota of a host (e.g., insect) and therebydecrease host fitness. The screening assays provided herein may beeffective to identify one or more modulating agents (e.g., phage) thattarget symbiotic microorganisms resident in the host and therebydecrease the fitness of the host. For example, the identified modulatingagent (e.g., phage) may be effective to decrease the viability ofpesticide- or allelochemical-degrading microorganisms (e.g., bacteriae.g., a bacterium that degrades a pesticide listed in Table 12), therebyincreasing the hosts sensitivity to a pesticide (e.g., sensitivity to apesticide listed in Table 12) or allelochemical agent.

For example, a phage library may be screened to identify a phage thattargets a specific endosymbiotic microorganism resident in a host. Insome instances, the phage library may be provided in the form of one ormore environmental samples (e.g., soil, pond sediments, or sewagewater). Alternatively, the phage library may be generated fromlaboratory isolates. The phage library may be co-cultured with a targetbacterial strain. After incubation with the bacterial strain, phage thatsuccessfully infect and lyse the target bacteria are enriched in theculture media. The phage-enriched culture may be sub-cultured withadditional bacteria any number of times to further enrich for phage ofinterest. The phage may be isolated for use as a modulating agent in anyof the methods or compositions described herein, wherein the phagealters the microbiota of the host in a manner that decreases hostfitness.

TABLE 12 Pesticides Aclonifen Fenchlorazole-ethyl PendimethalinAcetamiprid Fenothiocarb Penflufen Alanycarb Fenitrothion PenflufenAmidosulfuron Fenpropidin Pentachlorbenzene AminocyclopyrachlorFluazolate Penthiopyrad Amisulbrom Flufenoxuron PenthiopyradAnthraquinone Flu metralin Pirimiphos-methyl Asulam, sodium saltFluxapyroxad Prallethrin Benfuracarb Fuberidazole Profenofos BensulideGlufosinate-ammonium Proquinazid beta-HCH; beta-BCH GlyphosateProthiofos Bioresmethrin Group: Borax, Pyraclofos borate salts (seeBlasticidin-S Group: Paraffin Pyrazachlor oils, Mineral Borax; disodiumHalfenprox Pyrazophos tetraborate Boric acid Imiprothrin PyridabenBromoxynil heptanoate Imidacloprid Pyridalyl Bromoxynil octanoateIpconazole Pyridiphenthion Carbosulfan Isopyrazam PyrifenoxChlorantraniliprole Isopyrazam Quinmerac Chlordimeform Lenacil RotenoneChlorfluazuron Magnesium phosphide Sedaxane Chlorphropham MetaflumizoneSedaxane Climbazole Metazachlor Silafluofen Clopyralid MetazachlorSintofen Copper (II) hydroxide Metobromuron Spinetoram CyflufenamidMetoxuron Sulfoxaflor Cyhalothrin Metsulfuron-methyl TemephosCyhalothrin, gamma Milbemectin thiocloprid Decahydrate NaledThiamethoxam Diafenthiuron Napropamide Tolfenpyrad DimefuronNicosulfuron Tralomethrin Dimoxystrobin Nitenpyram Tributyltin compoundsDinotefuran Nitrobenzene Tridiphane Diquat dichloride o-phenylphenolTriflumizole Dithianon oils Validamycin E-Phosphamidon Oxadiargyl Zincphosphide EPTC Oxycarboxin Ethaboxam Paraffin oil Ethirimol Penconazole

EXAMPLES

The following is an example of the methods of the invention. It isunderstood that various other embodiments may be practiced, given thegeneral description provided above.

Example 1: Treatment of the Anopheles Mosquito with AzithromycinSolutions

This example demonstrates the ability to kill or decrease the fitness ofthe Anopheles coluzzii mosquitoes and decrease the transmission rate ofparasites by treatment with azithromycin, relatively broad but shallowantibacterial activity. It inhibits some Gram-positive bacteria, someGram-negative bacteria, and many atypical bacteria. The effect ofazithromycin on mosquitoes is mediated through the modulation ofbacterial populations endogenous to the mosquito that are sensitive toazithromycin. One targeted bacterial strain is Asaia.

The mosquito has been described as the most dangerous animal in theworld and malaria is one mosquito-borne disease that detrimentallyimpacts humans. There are about 3,500 mosquito species and those thattransmit malaria all belong to a sub-set called the Anopheles.Approximately 40 Anopheles species are able to transmit malaria thatsignificantly impacts human health.

Therapeutic Design:

Blood meals mixed with azithromycin solutions are formulated with finalantibiotic concentrations of 0 (negative control), 0.1, 1, or 5 μg/ml in1 mL of blood.

Experimental Design:

To prepare for the treatment, mosquitoes are grown in a lab environmentand medium. Experiments are performed with female mosquitoes from anAnopheles coluzzii Ngousso colony, originally established from fieldmosquitoes collected in Cameroon, maintained on human blood and fed asadults with 5% fructose. Larvae are fed tetramin fish food. Temperatureis maintained at 28° C. (±1° C.), 70-80% humidity on a 12 hr light/darkcycle.

Human Blood Feeding and Plasmodium Infections:

Plasmodium falciparum NF54 gametocytes are cultured in RPMI medium(GIBCO) including 300 mg. L-1 L-glutamine supplemented with 50 mg/Lhypoxanthine, 25 mM HEPES plus 10% heat-inactivated human serum withoutantibiotics. Two 25-mL cultures are started 17 and 14 days before theinfection at 0.5% parasitemia in 6% v/v washed O₊ red blood cells(RBCs). Media is changed daily. Before mosquito infection, centrifugedRBCs are pooled and supplemented with 20% fresh washed RBCs and humanserum (2:3 v/v ratio between RBCs and serum). Mosquitoes are offered ablood meal from a membrane-feeding device (2 ml Eppendorf tube) coveredwith Parafilm and kept at 37° C.

Azithromycin solutions are made by dissolving azithromycin(SIGMA-ALDRICH, PZ0007) in DMSO. Different volumes of azithromycinsolution are added to fresh blood to total 1 mL in preparation for bloodmeals. The final azithromycin concentrations in the blood are 0 (justsolvent as control solution), 0.1, 1, or 5 μg/ml.

For each Plasmodium infection, at least 100 age-matched, 2- to3-day-old, mosquitoes per condition are offered a control orexperimental blood meal from a membrane-feeding device (2 ml Eppendorftube) covered with parafilm and kept at 37° C. and nonengorgedmosquitoes are removed. Meals are given every four days for a total ofthree blood meals. Between the blood meals, mosquitoes are provided witha cotton pad moistened with distilled water for oviposition. Unfedmosquitoes are not removed after the second and later blood meals.Deaths are counted daily and carcasses are removed and stored for Asaiaanalysis as described herein. At least 50 mosquitoes per concentrationof azithromycin are used for each replicate. At the end of the lastblood meal, mosquitoes are kept for 12 hours before dissection.

Microbiota Analysis by Quantitative Polymerase Chain Reaction:

Before dissection, mosquitoes are immersed in 70% ethanol for 5 minutesthen rinsed 3 times in sterile phosphate-buffered saline (PBS) to killand remove surface bacteria, thus minimizing sample contamination withcuticle bacteria during dissection. The midgut of each mosquitoe(control and azithromycin treatment) is removed and frozen immediatelyon dry ice and stored at 20° C. until processing. Midguts are onlyexcluded from analysis if they burst and a substantial amount of the gutcontent is lost. Samples are homogenized in phenol-chloroform in aPrecellys 24 homogenizer (Bertin) using 0.5 mm-wide glass beads (Bertin)for 30 seconds at 6800 rpm and deoxy-ribonucleic acid (DNA) is extractedwith phenol-chloroform. The 16S ribosomal DNA (rDNA) is used for Asaiaquantification and is shown as a ratio of the Anopheles housekeepinggene 40S ribosomal protein S7 (Vector-Base gene ID AGAP010592). Primersequences for Asaia are: forward 5′-GTGCCGATCTCTAAAAGCCGTCTCA-3′ (SEQ IDNO: 219) and reverse 5′-TTCGCTCACCGGCTTCGGGT-3′ (SEQ ID NO: 220), andfor S7: forward 5′-GTGCGCGAGTTGGAGAAGA-3′ (SEQ ID NO: 221) and reverse5′-ATCGGTTTGGGCAGAATGC-3′ (SEQ ID NO: 222). Quantitative polymerasechain reaction (qPCR) is performed on a 7500 Fast Real-Time thermocycler(Applied Biosystems) using the SYBR Premix Ex Taq kit (Takara),following the manufacturer's instructions. Azithromycin treatedmosquitoes show a reduction of Asaia specific genes.

The survival rates of mosquitoes treated with azithromycin are comparedto the mosquitoes treated with the negative control. The survival rateof mosquitoes treated with azithromycin solution is decreased comparedto the control.

Example 2: Treatment of the Aedes vexans Mosquito with an AntibioticSolution

This Example demonstrates the ability to kill or decrease the fitness ofthe Aedes vexans mosquitoes by treatment with doxycycline, a broadspectrum antibiotic that inhibits protein production. The effect ofdoxycycline on mosquitoes is mediated through the modulation ofbacterial populations endogenous to the mosquito that are sensitive todoxycycline. One targeted bacterial strain is Wolbachia.

Successful control and eradication of porcine reproductive andrespiratory syndrome virus (PRRSV) is of great importance to the globalswine industry today. To reduce the risk of PRRSV entry, swine producersutilize stringent measures to enhance the biosecurity of their farms;however, infection of PRRSV in swine herds still frequently occurs. Onevector of transmission of PRRSV is the Aedes vexans mosquito. Aedesvexans is a cosmopolitan and common pest mosquito. On top of PRRSV, itis also a known vector of Dirofilaria immitis (dog heartworm);Myxomatosis (deadly rabbit virus disease) and Eastern equineencephalitis (deadly horse virus disease in the USA). Aedes vexans isthe most common mosquito in Europe, often composing more than 80% theEuropean mosquito community. Its abundance depends upon availability offloodwater pools. In summer, mosquito traps can collect up to 8,000mosquitoes per trap per night.

Therapeutic Design:

Blood meals mixed with doxycycline solutions are formulated with finalantibiotic concentrations of 0 (negative control), 1, 10, or 50 μg/ml in1 mL of blood

Experimental Design:

To prepare for the treatment, mosquitoes are grown in a lab environmentand medium. Experiments are performed with female mosquitoes from anAedes vexans, originally established from field mosquitoes collected ona field of the University of Minnesota St. Paul campus, maintained onhuman blood and fed as adults with 5% fructose. Doxycycline solutionsare made by dissolving doxycycline (SIGMA-ALDRICH, D9891) in sterilewater. Different volumes of a doxycycline solution are added to freshblood to total 1 mL in preparation for blood meals. The finaldoxycycline concentrations in the blood are approximately 0 (controlsolution), 1, 10 or 50 μg/ml.

For each replicate, age-matched, 2- to 3-day-old mosquitoes are offereda control or experimental blood meal from a membrane-feeding device (2ml Eppendorf tube) covered with parafilm and kept at 37° C. Nonengorgedmosquitoes are discarded. Meals are given every four days for a total ofthree blood meals. Between the blood meals, mosquitoes are provided witha cotton pad moistened with distilled water for oviposition. Unfedmosquitoes are not removed after the second and later blood meals.Deaths are counted daily and carcasses are removed and stored forWolbachia analysis as described herein. At least 50 mosquitoes perconcentration of doxycycline are used for each replicate. At the end ofthe last blood meal, mosquitoes are kept for 12 hours before dissection.

Microbiota Analysis by Quantitative Polymerase Chain Reaction:

Before dissection, mosquitoes are immersed in 70% ethanol for 5 minutesthen rinsed 3 times in sterile phosphate-buffered saline (PBS) to killand remove surface bacteria, thus minimizing sample contamination withcuticle bacteria during dissection. The midgut of each mosquito (controland doxycycline treatment) is removed and frozen immediately on dry iceand stored at 20° C. until processing. Midguts are only excluded fromanalysis if they burst and a substantial amount of the gut content islost. Samples are homogenized in phenol-chloroform in a Precellys 24homogenizer (Bertin) using 0.5 mm wide glass beads (Bertin) for 30seconds at 6800 rpm and deoxy-ribonucleic acid (DNA) is extracted withphenol-chloroform. The 16S ribosomal DNA (rDNA) is used for Wolbachiaquantification and is shown as a ratio of the Aedes housekeeping gene40S ribosomal protein S7 (Vector-Base gene ID AAEL009496). Primersequences for Wolbachia are: forward primer 5′-TCAGCCACACTGGAACTGAG-3′(SEQ ID NO: 225) and reverse primer 5′-TAACGCTAGCCCTCTCCGTA-3′ (SEQ IDNO: 226), and for S7: forward 5′-AAGGTCGACACCTTCACGTC-3′ (SEQ ID NO:227) and reverse 5′-CCGTTTGGTGAGGGTCTTTA-3′ (SEQ ID NO: 228).Quantitative polymerase chain reaction (qPCR) is performed on a 7500Fast Real-Time thermocycler (Applied Biosystems) using the SYBR PremixEx Taq kit (Takara), following the manufacturer's instructions.Doxycycline treated mosquitoes show a reduction of Wolbachia specificgenes.

The survival rates of mosquitoes treated with doxycycline solution arecompared to the mosquitoes treated with the negative control. Thesurvival rate of mosquitoes treated with doxycycline solution isdecreased compared to the control.

Example 3: Treatment of the Dermacentor andersoni, with an AntibioticSolution

This Example demonstrates the ability to kill or decrease the fitness ofthe tick, Dermacentor andersoni, by treatment with Liquamycin LA-200oxytetracycline, a broad spectrum antibiotic commonly used to treat abroad range of bacterial infections in cattle. The effect of LiquamycinLA-200 oxytetracycline on ticks is mediated through the modulation ofbacterial populations endogenous to the tick that are sensitive toLiquamycin LA-200 oxytetracycline. One targeted bacterial strain isRickettsia.

Ticks are obligate hematophagous arthropods that feed on vertebrates andcause great economic losses to livestock due to their ability totransmit diseases to humans and animals. In particular, ticks transmitpathogens throughout all continents and are labeled as principle vectorsof zoonotic pathogens. In fact, 415 new tick-borne bacterial pathogenshave been discovered since Lyme disease was characterized in 1982.Dermacentor andersoni, the Rocky Mountain wood tick, has been labeled a‘veritable Pandora's box of disease-producing agents’ and transmitsseveral pathogens, including Rickettsia rickettsii and Francisellatularensis. It is also a vector of Anaplasma marginale, the agent ofanaplasmosis, and the most widespread tick-borne pathogen of livestockworldwide (Gall et al., The ISME Journal 10:1846-1855, 2016). Economiclosses due to anaplasmosis in cattle are estimated to be $300 millionper year in the United States (Rochon et al., J. Med. Entomol.49:253-261, 2012).

Therapeutic design: A therapeutic dose (11 mg/kg of body weight) ofLiquamycin LA-200 oxytetracycline injection on −4, −1, +3 and +5 dayspost application of ticks.

Experimental Design:

Questing adult D. andersoni are collected by flag and drag techniques atsites in Burns, Oreg. and Lake Como, Mont. as described in (Scoles etal., J. Med. Entomol. 42:153-162, 2005). Field collected ticks are usedto establish laboratory colonies. For tick bacteria analysis, a cohortof adult F1 or F2 male ticks from each colony is fed on a Holstein calfand dissected to collect midguts (MG) and salivary glands (SG) forgenomic DNA isolation and bacteria quantification as follows:

A cohort of F1 ticks are fed on either antibiotic-treated calves oruntreated calves (control). The antibiotic-treated calves received atherapeutic dose (11 mg/kg of body weight) of Liquamycin LA-200oxytetracycline injections on −4, −1, +3 and +5 days post application ofticks, whereas untreated calves did not receive any injections(untreated control). Females ticks are allowed to oviposit to continue asecond generation of the untreated and treated ticks (F2 generation).The F2 treated generation arose from F1 adults that are exposed toantibiotics. The F2 ticks are not subjected to antibiotics.

F1 and F2 generation adult male ticks are fed for 7 days and thendissected within 24 h. Deaths are counted daily and ticks are removedand stored for Rickettsia analysis as described herein. Beforedissection, the ticks are surface sterilized and all dissection toolsare sterilized between each dissection. Tick MG and SG are dissected andpooled in groups of 30 with three biological replicates. Tissues arestored in Cell Lysis Solution (Qiagen, Valencia, Calif., USA) andProteinase K (1.25 mg/ml). Genomic DNA is isolated using the PureGeneExtraction kit (Qiagen) according to the manufacturer's specifications.

Quantitative analysis of Rickettsia bellii:

To quantify Rickettsia, rickA gene copy numbers are measured using SYBRGreen quantitative PCR of non-treated and antibiotic treated in F1 andF2 ticks. The quantity of Rickettsia is determined using Forward(5′-TACGCCACTCCCTGTGT CA-3′; SEQ ID NO: 229) and Reverse(5′-GATGTAACGGTATTAC ACCAACAG-3′; SEQ ID NO: 230) primers. The bacterialquantity is measured in F1 and F2 MG and SG of the pooled samples.Quantitative polymerase chain reaction (qPCR) is performed on a 7500Fast Real-Time thermocycler (Applied Biosystems) using the SYBR PremixEx Taq kit (Takara), following the manufacturer's instructions.Liquamycin LA-200 oxytetracycline treated ticks show a reduction ofRickettsia specific genes.

The survival rates of ticks treated with antibiotic solution arecompared to the ticks untreated. The survival rate of ticks treated withLiquamycin LA-200 oxytetracycline solution is decreased compared to theuntreated.

Example 4: Treatment of Mites that Infect Livestock with RifampicinSolutions

This Example demonstrates the ability to kill or decrease the fitness ofmites by treating them with an antibiotic solution. This Exampledemonstrates that the effect of oxytetracycline on mites is mediatedthrough the modulation of bacterial populations endogenous, such asBacillus, to the mites that are sensitive to oxytetracycline.

Sarcoptic mange is caused by mites that infest animals by burrowingdeeply into the skin and laying eggs inside the burrows. The eggs hatchinto the larval stage. The larval mites then leave the burrows, move upto the skin surface, and begin forming new burrows in healthy skintissue. Development from egg to adult is completed in about 2 weeks. Thelesions resulting from infestations by these mites are a consequence ofthe reaction of the animals' immune system to the mites' presence.Because of the intensity of the animals' immunological response, ittakes only a small number of mites to produce widespread lesions andgeneralized dermatitis. While mites can be killed with chemicallysynthesized miticides, these types of chemicals must sprayed on everyanimal in the herd with high-pressure hydraulic spray equipment toensure penetration by the spray into the skin. Furthermore, these typesof chemical pesticides may have detrimental ecological and/oragricultural effects.

Therapeutic Design:

Oxytetracycline solution is formulated with 0 (negative control), 1, 10,or 50 μg/ml in 10 mL of sterile water with 0.5% sucrose and essentialamino acids.

Experimental Design:

To determine whether adult mites at the reproductive stage havedifferent susceptibility compared to phoretic mites or their offspringbecause their cuticle is not hardened, mites living on livestock andmites associated with larvae and pupae are collected. This assay testsantibiotic solutions on different types of mites and determines howtheir fitness is altered by targeting endogenous microbes, such asBacillus.

The brood mites are collected from mite-infested pigs. Skin samples arecollected by gently scraping and lifting off encrusted areas from theinner ear area of the pig with a sharpened teaspoon and subsequentlyexamined for mites.

Mites are grouped per age and assayed separately. The age is determinedbased on the morphology and pigmentation of the larva or the pupa asfollows: mites collected from spinning larvae that are small enough tomove around are grouped into Group 1; mites collected from stretchedlarvae, which are too big to lay in the cell and start to stretchupright with their mouth in the direction of the cell opening, aregrouped into Group 2; and mites collected from pupae are grouped intoGroup 3. Mites are stored on their host larva or pupa in glass Petridishes until 50 units are collected. This ensures their feeding routineand physiological status remains unchanged. To prevent mites fromstraying from their host larva or pupa or climbing onto one another,only hosts at the same development stage are kept in the same dish.

The equipment—a stainless steel ring (56 mm inner diameter, 2-3 mmheight) and 2 glass circles (62 mm diameter)—is cleaned with acetone andhexane or pentane to form the testing arena. The oxytetracyclinesolutions and control solution are applied on the equipment by sprayingthe glass disks and ring of the arena homogeneously. For this, areservoir is loaded with 1 ml of the solutions; the distance of thesprayed surface from the bottom end of the tube is set at 11 mm and a0.0275 inch nozzle is used. The pressure is adjusted (usually in therange 350-500 hPa) until the amount of solution deposited is 1±0.05mg/cm2. The antibiotic solutions are poured in their respective dishes,covering the whole bottom of the dishes, and residual liquid isevaporated under a fume hood. The ring is placed between the glasscircles to build a cage. The cages are used within 60 hr of preparation,for not more than three assays, in order to control the exposure ofmites to antibiotic solutions. 10 to 15 mites are introduced in thiscage and the equipment pieces are bound together with droplets of meltedwax. Mites collected from spinning larvae, stretched larvae, white eyedpupae and dark eyed with white and pale body are used.

After 4 hours, mites are transferred into a clean glass Petri dish (60mm diameter) with two or three white eye pupae (4-5 days after capping)to feed on. The mites are observed under a dissecting microscope at 4hr, 24 hr, and 48 hr after being treated with the antibiotic or thecontrol solutions, and classified according to the following categories:

-   -   Mobile: they walk around when on their legs, whether after being        poked by a needle.    -   Paralyzed: they move one or more appendages, unstimulated or        after stimulation, but they cannot move around.    -   Dead: immobile and do not react to 3 subsequent stimulations.

A sterile toothpick or needle is used to stimulate the mites by touchingtheir legs. New tooth picks or sterile needles are used for stimulatingeach group to avoid contamination between mite groups.

The assays are carried out at 32.5° C. and 60-70% relative humidity. Ifthe mortality in the controls exceeds 30%, the replicate is excluded.Each experiment is replicated with four series of cages.

The status of Bacillus in mite groups is assessed by PCR. Total DNA isisolated from control (non-oxytetracycline treated) and oxytetracylinetreated individuals (whole body) using a DNA Kit (OMEGA, Bio-tek)according to the manufacturer's protocol. The primers for Bacillus,forward primer 5′-GAGGTAGACGAAGCGACCTG-3′ (SEQ ID NO: 231) and reverseprimer 5′-TTCCCTCACGGTACTGGTTC-3′ (SEQ ID NO: 232), are designed basedon 23S-5S rRNA sequences obtained from the Bacillus genome (AccessionNumber: AP007209.1) (Takeno et al., J. Bacteriol. 194(17):4767-4768,2012) using Primer 5.0 software (Primer-E Ltd., Plymouth, UK). The PCRamplification cycles included an initial denaturation step at 95° C. for5 min, 35 cycles of 95° C. for 1 min, 59° C. for 1 min, and 72° C. for 2min, and a final extension step of 5 min at 72° C. Amplificationproducts from oxytetracyline treated and control samples are analyzed on1% agarose gels, stained with SYBR safe, and visualized using an imagingSystem.

The survival rates of mites treated with an oxytetracyline solution arecompared to the Varroa mites treated with the negative control.

The survival rate and the mobility of mites treated with oxytetracylinesolution are expected to be decreased compared to the control.

Example 5: Production of a Phage Library

This Example demonstrates the acquisition of a phage collection fromenvironmental samples.

Therapeutic design: Phage library collection having the following phagefamilies: Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridae,Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae,Corticoviridae, Cystoviridae, Fuselloviridae, Gluboloviridae,Guttaviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae,Tectiviridae

Experimental Design:

Multiple environmental samples (soil, pond sediments, sewage water) arecollected in sterile 1 L flasks over a period of 2 weeks and areimmediately processed as described below after collection and storedthereafter at 4° C. Solid samples are homogenized in steriledouble-strength difco luria broth (LB) or tryptic soy broth (TSB)supplemented with 2 mM CaCl2) to a final volume of 100 mL. The pH andphosphate levels are measured using phosphate test strips. Forpurification, all samples are centrifuged at 3000-6000 g in a Megafuge1.0R, Heraeus, or in Eppendorf centrifuge 5702 R, for 10-15 min at +4°C., and filtered through 0.2-μm low protein filters to remove allremaining bacterial cells. The supernatant is stored at 4° C. in thepresence of chloroform in a glass bottle.

Example 6: Identification of Target Specific Phage

This Example demonstrates the isolation, purification, andidentification of single target specific phages from a heterogeneousphage library.

Experimental Design:

20-30 ml of the phage library described in Example 5 is diluted to avolume of 30-40 ml with LB-broth. The target bacterial strain, e.g.,Buchnera, is added (50-200 μl overnight culture grown in LB-broth) toenrich phages that target this specific bacterial strain in the culture.This culture is incubated overnight at +37° C., shaken at 230 rpm.Bacteria from this enrichment culture are removed by centrifugation(3000-6000 g in Megafuge 1.0R, Heraeus, or in Eppendorf centrifuge 5702R, 15-20 min, +4° C.) and filtered (0.2 or 0.45 μm filter). 2.5 ml ofthe bacteria free culture is added to 2.5 ml of LB-broth and 50-100 μlof the target bacteria to enrich the phages. The enrichment culture isgrown overnight as above. A sample from this enrichment culture iscentrifuged at 13,000 g for 15 min at room temperature and 10 μl of thesupernatant is plated on a LB-agar containing petri dish along with100-300 μl of the target bacteria and 3 ml of melted 0.7% soft-agar. Theplates are incubated overnight at +37° C. Each of the plaques observedon the bacterial lawn are picked and transferred into 500 μl ofLB-broth. A sample from this plaque-stock is further plated on thetarget bacteria. Plaque-purification is performed three times for alldiscovered phages in order to isolate a single homogenous phage from theheterogeneous phage mix.

Lysates from plates with high-titer phages (>1×10{circumflex over ( )}10PFU/ml) are prepared by harvesting overlay plates of a host bacteriumstrain exhibiting confluent lysis. After being flooded with 5 ml ofbuffer, the soft agar overlay is macerated, clarified by centrifugation,and filter sterilized. The resulting lysates are stored at 4° C.High-titer phage lysates are further purified by isopycnic CsClcentrifugation, as described in (Summer et al., J. Bacteriol.192:179-190, 2010).

DNA is isolated from CsCl-purified phage suspensions as described in(Summer, Methods Mol. Biol. 502:27-46, 2009). An individual isolatedphage is sequenced as part of two pools of phage genomes by using a 454pyrosequencing method. Phage genomic DNA is mixed in equimolar amountsto a final concentration of about 100 ng/L. The pooled DNA is sheared,ligated with a multiplex identifier (MID) tag specific for each of thepools, and sequenced by pyrosequencing using a full-plate reaction on aRoche FLX Titanium sequencer according to the manufacturer's protocols.The pooled phage DNA is present in two sequencing reactions. The trimmedFLX Titanium flow-gram output corresponding to each of the pools isassembled individually by using Newbler Assembler version 2.5.3 (454Life Sciences), by adjusting the settings to include only readscontaining a single MID per assembly. The identity of individual contigsis determined by PCR using primers generated against contig sequencesand individual phage genomic DNA preparations as the template.Sequencher 4.8 (Gene Codes Corporation) is used for sequence assemblyand editing. Phage chromosomal end structures are determinedexperimentally. Cohesive (cos) ends for phages are determined bysequencing off the ends of the phage genome and sequencing the PCRproducts derived by amplification through the ligated junction ofcircularized genomic DNA, as described in (Summer, Methods Mol. Biol.502:27-46, 2009). Protein-coding regions are initially predicted usingGeneMark.hmm (Lukashin et al. Nucleic Acids Res. 26:1107-1115, 1998),refined through manual analysis in Artemis (Rutherford et al.,Bioinformatics 16:944-945, 2000.), and analyzed through the use of BLAST(E value cutoff of 0.005) (Camacho et al., BMC Bioinformatics 10:421,2009). Proteins of particular interest are additionally analyzed byInterProScan (Hunter et al., Nucleic Acids Res. 40:D306-D312, 2012).

Electron microscopy of CsCl-purified phage (>1×10{circumflex over ( )}11PFU/ml) that lysed the endosymbiotic bacteria, Buchnera, is performed bydiluting stock with the tryptic soy broth buffer. Phages are appliedonto thin 400-mesh carbon-coated Formvar grids, stained with 2% (wt/vol)uranyl acetate, and air dried. Specimens are observed on a JEOL 1200EXtransmission electron microscope operating at an acceleration voltage of100 kV. Five virions of each phage are measured to calculate mean valuesand standard deviations for dimensions of capsid and tail, whereappropriate.

Example 7: Treatment of Aphids with a Solution of Purified Phages

This Example demonstrates the ability to kill or decrease the fitness ofaphids by treating them with a phage solution. This Example demonstratesthat the effect of phage on aphids is mediated through the modulation ofbacterial populations endogenous to the aphid that are sensitive tophages. One targeted bacterial strain is Buchnera with the phageidentified in Example 6.

Aphids are representative species for testing microbiota modulatingagents and effects on fitness of the aphids.

Therapeutic Design:

Phage solutions are formulated with 0 (negative control), 10², 10⁵, or10⁸ plaque-forming units (pfu)/ml phage from Example 6 in 10 mL ofsterile water with 0.5% sucrose and essential amino acids.

Experimental Design:

To prepare for the treatment, aphids are grown in a lab environment andmedium. In a climate-controlled room (16 h light photoperiod; 60±5% RH;20±2° C.), fava bean plants are grown in a mixture of vermiculite andperlite at 24° C. with 16 h of light and 8 h of darkness. To limitmaternal effects or health differences between plants, 5-10 adults fromdifferent plants are distributed among 10 two-week-old plants, andallowed to multiply to high density for 5-7 days. For experiments,second and third instar aphids are collected from healthy plants anddivided into treatments so that each treatment receives approximatelythe same number of individuals from each of the collection plants.

Phage solutions are prepared as described herein. Wells of a 96-wellplate are filled with 200 μl of artificial aphid diet (Febvay et al.,Canadian Journal of Zoology 66(11):2449-2453, 1988) and the plate iscovered with parafilm to make a feeding sachet. Artificial diet iseither mixed with sterile water and with 0.5% sucrose and essentialamino acids as a negative control or phage solutions with varyingconcentrations of phages. Phage solutions are mixed with artificial dietto get final concentrations of phages between 10² to 10⁸ (pfu)/ml.

For each replicate treatment, 30-50 second and third instar aphids areplaced individually in wells of a 96-well plate and a feeding sachetplate is inverted above them, allowing the insects to feed through theparafilm and keeping them restricted to individual wells. Experimentalaphids are kept under the same environmental conditions as aphidcolonies. After the aphids are fed for 24 hr, the feeding sachet isreplaced with a new one containing sterile artificial diet and a newsterile sachet is provided every 24 h for 4 days. At the time that thesachet is replaced, aphids are also checked for mortality. An aphid iscounted as dead if it had turned brown or is at the bottom of the welland does not move during the observation. If an aphid is on the parafilmof the feeding sachet but not moving, it is assumed to be feeding andalive.

The status of Buchnera in aphid samples is assessed by PCR. Aphidsadults from the negative control (non-phage treated) and phage treatedgroups are first surface-sterilized with 70% ethanol for 1 min, 10%bleach for 1 min and three washes of ultrapure water for 1 min. TotalDNA is extracted from each individual (whole body) using an Insect DNAKit (OMEGA, Bio-tek) according to the manufacturer's protocol. Theprimers for Buchnera, forward primer 5′-GTCGGCTCATCACATCC-3′ (SEQ ID NO:233) and reverse primer 5′-TTCCGTCTGTATTATCTCCT-3′ (SEQ ID NO: 234), aredesigned based on 23S-5S rRNA sequences obtained from the Buchneragenome (Accession Number: GCA_000009605.1) (Shigenobu et al., Nature407:81-86, 2000) using Primer 5.0 software (Primer-E Ltd., Plymouth,UK). The PCR amplification cycles included an initial denaturation stepat 95° C. for 5 min, 35 cycles of 95° C. for 30 s, 55° C. for 30 s, and72° C. for 60 s, and a final extension step of 10 min at 72° C.Amplification products from rifampicin treated and control samples areanalyzed on 1% agarose gels, stained with SYBR safe, and visualizedusing an imaging System. Phage treated aphids show a reduction ofBuchnera specific genes.

The survival rates of aphids treated with Buchnera specific phages arecompared to the aphids treated with the negative control. The survivalrate of aphids treated with Buchnera specific phages is decreased ascompared to the control treated aphids.

Example 8: Production of a colA Bacteriocin Solution

This Example demonstrates the production and purification of colAbacteriocin.

Construct Sequence:

(SEQ ID NO: 235) catatgatgacccgcaccatgctgtttctggcgtgcgtggcggcgctgtatgtgtgcattagcgcgaccgcgggcaaaccggaagaatttgcgaaactgagcgatgaagcgccgagcaacgatcaggcgatgtatgaaagcattcagcgctatcgccgctttgtggatggcaaccgctataacggcggccagcagcagcagcagcagccgaaacagtgggaagtgcgcccggatctgagccgcgatcagcgcggcaacaccaaagcgcaggtggaaattaacaaaaaaggcgataaccatgatattaacgcgggctggggcaaaaacattaacggcccggatagccataaagatacctggcatgtgggcggcagcgtgcgctggctcgag

Experimental Design:

DNA is generated by PCR with specific primers with upstream (NdeI) anddownstream (XhoI) restriction sites. Forward primerGTATCTATTCCCGTCTACGAACATATGGAATTCC (SEQ ID NO: 236) and reverse primerCCGCTCGAGCCATCTGACACTTCCTCCAA (SEQ ID NO: 237). Purified PCR fragments(Nucleospin Extract II-Macherey Nagel) are digested with NdeI or XhoIand then the fragments are ligated. For colA cloning, the ligated DNAfragment is cloned into pcr2.1 (GenBank database accession numberEY122872) vector (Anselme et al., BMC Biol. 6:43, 2008). The nucleotidesequence is systematically checked (Cogenics).

The plasmid with colA sequence is expressed in BL21 (DE3)/pLys. Bacteriaare grown in LB broth at 30° C. At an OD600 of 0.9, isopropylβ-D-1-thiogalactopyranoside (IPTG) is added to a final concentration of1 mM and cells were grown for 6 h. Bacteria are lysed by sonication in100 mM NaCL, 1% Triton X-100, 100 mM Tris-base pH 9.5, and proteins areloaded onto a HisTrap HP column (GE Healthcare). The column is washedsuccessively with 100 mM NaCl, 100 mM Tris-HCl pH 6.8, and PBS. Elutionis performed with 0.3M imidazol in PBS. Desalting PD-10 columns (GEHealthcare) are used to eliminate imidazol and PBS solubilized peptidesare concentrated on centrifugal filter units (Millipore).

ColA Protein Sequence:

(SEQ ID NO: 211) MTRTMLFLAC VAALYVCISA TAGKPEEFAK LSDEAPSNDQAMYESIQRYR RFVDGNRYNG GQQQQQQPKQ WEVRPDLSRDQRGNTKAQVE INKKGDNHDI NAGWGKNING PDSHKDTWHV GGSVRW

Example 9: Treatment of Aphids with a Solution of colA Bacteriocin

This Example demonstrates the ability to kill or decrease the fitness ofaphids by treating them with a bacteriocin solution. This Exampledemonstrates that the effect of bacteriocins on aphids is mediatedthrough the modulation of bacterial populations endogenous to the aphidthat are sensitive to ColA bacteriocin. One targeted bacterial strain isBuchnera with the bacteriocin produced in Example 8.

Therapeutic Design:

ColA solutions are formulated with 0 (negative control), 0.6, 1, 50 or100 mg/ml of ColA from Example 8 in 10 mL of sterile water with 0.5%sucrose and essential amino acids.

Experimental Design:

To prepare for the treatment, aphids are grown in a lab environment andmedium. In a climate-controlled room (16 h light photoperiod; 60±5% RH;20±2° C.), plants are grown in a mixture of vermiculite and perlite andare infested with aphids. In the same climatic conditions, E. balteatuslarvae are obtained from a mass production; the hoverflies are rearedwith sugar, pollen, and water; and the oviposition is induced by theintroduction of infested host plants in the rearing cage during 3 h. Thecomplete life cycle takes place on the host plants that are dailyre-infested with aphids.

Wells of a 96-well plate are filled with 200 μl of artificial aphid diet(Febvay et al., Canadian Journal of Zoology 66(11):2449-2453, 1988) andthe plate is covered with parafilm to make a feeding sachet. Artificialdiet is either mixed with the solution of sterile water with 0.5%sucrose and essential amino acids as a negative control or ColAsolutions with varying concentrations of ColA. ColA solutions are mixedwith artificial diet to obtain final concentrations between 0.6 to 100mg/ml.

For each replicate treatment, 30-50 second and third instar aphids areplaced individually in wells of a 96-well plate and a feeding sachetplate is inverted above them, allowing the insects to feed through theparafilm and keeping them restricted to individual wells. Experimentalaphids are kept under the same environmental conditions as aphidcolonies. After the aphids are fed for 24 hr, the feeding sachet isreplaced with a new one containing sterile artificial diet and a newsterile sachet is provided every 24 h for 4 days. At the time that thesachet is replaced, aphids are also checked for mortality. An aphid iscounted as dead if it had turned brown or is at the bottom of the welland does not move during the observation. If an aphid is on the parafilmof the feeding sachet but not moving, it is assumed to be feeding andalive.

The status of Buchnera in aphid samples is assessed by PCR. Aphidsadults from the negative control and phage treated are firstsurface-sterilized with 70% ethanol for 1 min, 10% bleach for 1 min andthree washes of ultrapure water for 1 min. Total DNA is extracted fromeach individual (whole body) using an Insect DNA Kit (OMEGA, Bio-tek)according to the manufacturer's protocol. The primers for Buchnera,forward primer 5′-GTCGGCTCATCACATCC-3′ (SEQ ID NO: 233) and reverseprimer 5′-TTCCGTCTGTATTATCTCCT-3′ (SEQ ID NO: 234), are designed basedon 23S-5S rRNA sequences obtained from the Buchnera genome (AccessionNumber: GCA_000009605.1) (Shigenobu, et al., Nature 200.407, 81-86)using Primer 5.0 software (Primer-E Ltd., Plymouth, UK). The PCRamplification cycles included an initial denaturation step at 95° C. for5 min, 35 cycles of 95° C. for 30 s, 55° C. for 30 s, and 72° C. for 60s, and a final extension step of 10 min at 72° C. Amplification productsfrom rifampicin treated and control samples are analyzed on 1% agarosegels, stained with SYBR safe, and visualized using an imaging System.ColA treated aphids show a reduction of Buchnera specific genes.

The survival rates of aphids treated with Buchnera specific ColAbacteriocin are compared to the aphids treated with the negativecontrol. The survival rate of aphids treated with Buchnera specific ColAbacteriocin is decreased as compared to the control treated aphids.

Example 10: Treatment of Aphids with Rifampicin Solutions

This Example demonstrates the ability to kill or decrease the fitness ofaphids by treating them with rifampicin, a narrow spectrum antibioticthat inhibits DNA-dependent RNA synthesis by inhibiting a bacterial RNApolymerase. This Example demonstrates that the effect of rifampicin onaphids is mediated through the modulation of bacterial populationsendogenous to the aphid that are sensitive to rifampicin. One targetedbacterial strain is Buchnera.

Therapeutic Design:

The antibiotic solutions are formulated with 0 (negative control), 1,10, or 50 μg/ml of rifampicin in 10 mL of sterile water with 0.5%sucrose and essential amino acids.

Experimental Design:

To prepare for the treatment, aphids are grown in a lab environment andmedium. In a climate-controlled room (16 h light photoperiod; 60±5% RH;20±2° C.), fava bean plants are grown in a mixture of vermiculite andperlite at 24° C. with 16 h of light and 8 h of darkness. To limitmaternal effects or health differences between plants, 5-10 adults fromdifferent plants are distributed among 10 two-week-old plants, andallowed to multiply to high density for 5-7 days. For experiments,second and third instar aphids are collected from healthy plants anddivided into treatments so that each treatment receives approximatelythe same number of individuals from each of the collection plants.

Rifampicin solutions are made by dissolving rifampicin (SIGMA-ALDRICH,557303) in sterile water with 0.5% sucrose and essential aminoacids.Wells of a 96-well plate are filled with 200 μl of artificial aphid diet(Febvay et al., Canadian Journal of Zoology 66(11):2449-2453, 1988) andthe plate is covered with parafilm to make a feeding sachet. Artificialdiet is either mixed with sterile water and with 0.5% sucrose andessential aminoacids as a negative control or a rifampicin solution withone of the concentrations of rifampicin. Rifampicin solutions are mixedwith artificial diet to get final concentrations of the antibioticbetween 1 and 50 μg/mL.

For each replicate treatment, 30-50 second and third instar aphids areplaced individually in wells of a 96-well plate and a feeding sachetplate is inverted above them, allowing the insects to feed through theparafilm and keeping them restricted to individual wells. Experimentalaphids are kept under the same environmental conditions as aphidcolonies. After the aphids are fed for 24 hr, the feeding sachet isreplaced with a new one containing sterile artificial diet and a newsterile sachet is provided every 24 h for four days. At the time thatthe sachet is replaced, aphids are also checked for mortality. An aphidis counted as dead if it had turned brown or is at the bottom of thewell and does not move during the observation. If an aphid is on theparafilm of the feeding sachet but not moving, it is assumed to befeeding and alive.

The status of Buchnera in aphid samples is assessed by PCR. Total DNA isisolated from control (non-rifampicin treated) and rifampicin treatedindividuals using an Insect DNA Kit (OMEGA, Bio-tek) according to themanufacturer's protocol. The primers for Buchnera, forward primer5′-GTCGGCTCATCACATCC-3′ (SEQ ID NO: 233) and reverse primer5′-TTCCGTCTGTATTATCTCCT-3′ (SEQ ID NO: 234), are designed based on23S-5S rRNA sequences obtained from the Buchnera genome (AccessionNumber: GCA_000009605.1) (Shigenobu et al., Nature 407:81-86, 2000)using Primer 5.0 software (Primer-E Ltd., Plymouth, UK). The PCRamplification cycles included an initial denaturation step at 95° C. for5 min, 35 cycles of 95° C. for 30 s, 55° C. for 30 s, and 72° C. for 60s, and a final extension step of 10 min at 72° C. Amplification productsfrom rifampicin treated and control samples are analyzed on 1% agarosegels, stained with SYBR safe, and visualized using an imaging System.Rifampicin treated aphids show a reduction of Buchnera specific genes.

The survival rates of aphids treated with rifampicin solution arecompared to the aphids treated with the negative control. The survivalrate of aphids treated with rifampicin solution is decreased compared tothe control.

Example 11: High Throughput Screening (HTS) for Buchnera TargetingMolecules

This Example demonstrates the identification of molecules that targetBuchnera.

Experimental Design:

A HTS to identify inhibitors of targeted bacterial strains, Buchnera,uses sucrose fermentation in pH-MMSuc medium (Ymele-Leki et al., PLoSONE 7(2):e31307, 2012) to decrease the pH of the medium. pH indicatorsin the medium monitor medium acidification spectrophotometricallythrough a change in absorbance at 615 nm (A615). A target bacterialstrain, Buchnera, derived from a glycerol stock, is plated on an LB-agarplate and incubated overnight at 37° C. A loopful of cells is harvested,washed three times with PBS, and then resuspended in PBS at an opticaldensity of 0.015.

For the HTS, 10 μL of this bacterial cell suspension is aliquoted intothe wells of a 384-well plate containing 30 μL of pH-MMSuc medium and100 nL of a test compound fraction from a natural product library ofpre-fractionated extracts (39,314 extracts arrayed in 384-well plates)from microbial sources, such as fungal endophytes, bacterial endophytes,soil bacteria, and marine bacteria, described in (Ymele-Leki et al.,PLoS ONE 7(2):e31307, 2012). For each assay, the A615 is measured afterincubation at room temperature at 6 hr and 20 hr. This step is automatedand validated in the 384-well plate format using an EnVision™ multi-wellspectrophotometer to test all fractions from the library. Fractions thatdemonstrate delayed medium acidification by sucrose fermentation andinhibited cell growth are selected for further purification andidentification.

Example 12: Isolation and Identification of Buchnera Specific Molecules

This Example demonstrates the isolation and identification of an isolatefrom the fraction described in Example 11 that blocks sucrosefermentation and inhibits cell growth of Buchnera.

Experimental Design:

The fraction described in Example 11 is resuspended in 90%water/methanol and passed over a C18 SPE column to get fraction I. Thecolumn is then washed with methanol to get fraction II. Fraction II isseparated on an Agilent 1100 series HPLC with a preparative Phenyl-hexylcolumn (Phenomenex, Luna, 25 cm610 mm, 5 mm particle size) using anelution buffer with 20% acetonitrile/water with 0.1% formic acid at aflow rate of 2 mL/min for 50 minutes. This yields multiple compounds atdifferent elution times. Spectra for each compound is obtained on anAlpha FT-IR mass spectrometer (Bruker), an Ultrospec™ 5300 proUV/Visible Spectrophotometer (Amersham Biosciences), and an INOVA 600MHz nuclear magnetic resonance spectrometer (Varian).

Example 13: Treatment of Aphids with a Solution of a Buchnera SpecificMolecule

This Example demonstrates the ability to kill or decrease the fitness ofaphids by treating them with one of the compounds identified in Example12 through the modulation of bacterial populations endogenous to theaphid that are sensitive to this compound. One targeted bacterial strainis Buchnera.

Therapeutic Design:

Each compound from Example 12 is formulated at 0 (negative control),0.6, 1, 20 or 80 μg/ml in 10 mL of sterile water with 0.5% sucrose andessential amino acids.

Experimental Design:

To prepare for the treatment, aphids are grown in a lab environment andmedium. In a climate-controlled room (16 h light photoperiod; 60±5% RH;20±2° C.), fava bean plants are grown in a mixture of vermiculite andperlite at 24° C. with 16 h of light and 8 h of darkness. To limitmaternal effects or health differences between plants, 5-10 adults fromdifferent plants are distributed among 10 two-week-old plants, andallowed to multiply to high density for 5-7 days. For experiments,second and third instar aphids are collected from healthy plants anddivided into treatments so that each treatment receives approximatelythe same number of individuals from each of the collection plants.

Wells of a 96-well plate are filled with 200 μl of artificial aphid diet(Febvay et al., Canadian Journal of Zoology 66(11):2449-2453, 1988) andthe plate is covered with parafilm to make a feeding sachet. Artificialdiet is either mixed with sterile water with 0.5% sucrose and essentialamino acids as a negative control or solutions with varyingconcentrations of the compound.

For each replicate treatment, 30-50 second and third instar aphids areplaced individually in wells of a 96-well plate and a feeding sachetplate is inverted above them, allowing the insects to feed through theparafilm and keeping them restricted to individual wells. Experimentalaphids are kept under the same environmental conditions as aphidcolonies. After the aphids are fed for 24 hr, the feeding sachet isreplaced with a new one containing sterile artificial diet and a newsterile sachet is provided every 24 h for 4 days. At the time that thesachet is replaced, aphids are also checked for mortality. An aphid iscounted as dead if it had turned brown or is at the bottom of the welland does not move during the observation. If an aphid is on the parafilmof the feeding sachet but not moving, it is assumed to be feeding andalive.

The status of Buchnera in aphid samples is assessed by PCR. Aphids fromthe negative control and compound 1 treated are first surface-sterilizedwith 70% ethanol for 1 min, 10% bleach for 1 min and three washes ofultrapure water for 1 min. Total DNA is extracted from each individual(whole body) using an Insect DNA Kit (OMEGA, Bio-tek) according to themanufacturer's protocol. The primers for Buchnera, forward primer5′-GTCGGCTCATCACATCC-3′ (SEQ ID NO: 233) and reverse primer5′-TTCCGTCTGTATTATCTCCT-3′ (SEQ ID NO: 234), are designed based on23S-5S rRNA sequences obtained from the Buchnera genome (AccessionNumber: GCA_000009605.1) (Shigenobu et al., Nature 407:81-86, 2000)using Primer 5.0 software (Primer-E Ltd., Plymouth, UK). The PCRamplification cycles included an initial denaturation step at 95° C. for5 min, 35 cycles of 95° C. for 30 s, 55° C. for 30 s, and 72° C. for 60s, and a final extension step of 10 min at 72° C. Amplification productsfrom compound 1 treated and control samples are analyzed on 1% agarosegels, stained with SYBR safe, and visualized using an imaging System.Reduction of Buchnera specific genes indicates antimicrobial activity ofcompound 1.

The survival rate of aphids treated with the compound is compared to theaphids treated with the negative control. A decrease in the survivalrate of aphids treated with the compound is expected to indicateantimicrobial activity of the compound.

Example 14: Insects Treated with an Antibiotic Solution

This Example demonstrates the treatment of aphids with rifampicin, anarrow spectrum antibiotic that inhibits DNA-dependent RNA synthesis byinhibiting a bacterial RNA polymerase. This Example demonstrates thatthe effect of rifampicin on a model insect species, aphids, was mediatedthrough the modulation of bacterial populations endogenous to the insectthat were sensitive to rifampicin. One targeted bacterial strain isBuchnera.

Therapeutic Design

The antibiotic solution was formulated according to the means ofdelivery, as follows (FIG. 1A-1G):

1) Through the plants: with 0 (negative control) or 100 μg/ml ofrifampicin formulated in an artificial diet (based on Akey and Beck,1971; see Experimental Design) with and without essential amino acids (2mg/ml histidine, 2 mg/ml isoleucine, 2 mg/ml leucine, 2 mg/ml lysine, 1mg/ml methionine, 1.62 mg/ml phenylalanine, 2 mg/ml threonine, 1 mg/mltryptophan, and 2 mg/ml valine).

2) Leaf coating: 100 μl of 0.025% nonionic organosilicone surfactantsolvent Silwet L-77 in water (negative control) or 100 μl of 50 μg/ml ofrifampicin formulated in solvent solution was applied directly to theleaf surface and allowed to dry.

3) Microinjection: injection solutions were either 0.025% nonionicorganosilicone surfactant solvent Silwet L-77 in water (negativecontrol), or 50 μg/ml of rifampicin formulated in solvent solution.

4) Topical delivery: 100 μl of 0.025% nonionic organosilicone surfactantsolvent Silwet L-77 (negative control), or 50 μg/ml of rifampicinformulated in solvent solution were sprayed using a 30 mL spray bottle.

5) Leaf injection method A—Leaf perfusion and cutting: leaves wereinjected with approximately 1 ml of 50 μg/ml of rifampicin in water withfood coloring or approximately 1 ml of negative control with water andfood coloring. Leaves were cut into 2×2 cm squared pieces and aphidswere placed on the leaf pieces.

6) Leaf perfusion and delivery through plant: Leaves were injected withapproximately 1 ml of 100 μg/ml of rifampicin in water plus foodcoloring or approximately 1 ml of negative control with water and foodcoloring. The stem of injected leaf was then placed into an Eppendorftube with 1 ml of 100 μg/ml of rifampicin plus water and food coloringor 1 ml of negative control with only water and food coloring.

7) Combination delivery method: a) Topical delivery to aphid and plant:via spraying both aphids and plants with 0.025% nonionic organosiliconesurfactant solvent Silwet L-77 in water (negative control) or 100 μg/mlof rifampicin formulated in solvent solution using a 30 mL, b) Deliverythrough plant: water only (negative control) or 100 μg/ml of rifampicinformulated in water.

Plant Delivery Experimental Design:

Aphids (LSR-1 strain, Acyrthosiphon pisum) were grown on fava beanplants (Vroma Vicia faba from Johnny's Selected Seeds) in aclimate-controlled incubator (16 h light/8 h dark photoperiod; 60±5% RH;25±2° C.). Prior to being used for aphid rearing, fava bean plants weregrown in potting soil at 24° C. with 16 h of light and 8 h of darkness.To limit maternal effects or health differences between plants, 5-10adults from different plants were distributed among 10 two-week-oldplants, and allowed to multiply to high density for 5-7 days. Forexperiments, first instar aphids were collected from healthy plants anddivided into 3 different treatment groups: 1) artificial diet alonewithout essential amino acids, 2) artificial diet alone withoutessential amino acids and 100 μg/ml rifampicin, and 3) artificial dietwith essential amino acids and 100 μg/ml rifampicin). Each treatmentgroup received approximately the same number of individuals from each ofthe collection plants.

The artificial diet used was made as previously published (Akey andBeck, 1971 Continuous Rearing of the Pea Aphid, Acyrthosiphon pisum, ona Holidic Diet), with and without the essential amino acids (2 mg/mlhistidine, 2 mg/ml isoleucine, 2 mg/ml leucine, 2 mg/ml lysine, 1 mg/mlmethionine, 1.62 mg/ml phenylalanine, 2 mg/ml threonine, 1 mg/mltryptophan, and 2 mg/ml valine), except neither diet included homoserineor beta-alanyltyrosine. The pH of the diets was adjusted to 7.5 with KOHand diets were filter sterilized through a 0.22 μm filter and stored at4° C. for short term (<7 days) or at −80° C. for long term.

Rifampicin (Tokyo Chemical Industry, LTD) stock solutions were made at25 mg/ml in methanol, sterilized by passing through a 0.22 μm syringefilter, and stored at −20° C. For treatments (see Therapeutic design),the appropriate amount of stock solution was added to the artificialdiet with or without essential amino acids to obtain a finalconcentration of 100 μg/ml rifampicin. The diet was then placed into a1.5 ml Eppendorf tube with a fava bean stem with a leaf and the openingof the tube was closed using parafilm. This artificial diet feedingsystem was then placed into a deep petri dish (Fisher Scientific, Cat#FB0875711) and aphids were applied to the leaves of the plant.

For each treatment, 33 aphids were placed onto each leaf. Artificialdiet feeding systems were changed every 2-3 days throughout theexperiment. Aphids were monitored daily for survival and dead aphidswere removed from the deep petri dish housing the artificial feedingsystem when they were discovered.

In addition, the developmental stage (1^(st), 2^(nd), 3^(rd), 4^(th),5^(th) instar) was determined daily throughout the experiment. Once anaphid reached the 4th instar stage, they were given their own artificialfeeding system in a deep petri dish so that fecundity could be monitoredonce they reached adulthood.

For adult aphids, new nymphs were counted daily and then discarded. Atthe end of the experiments, fecundity was determined as the mean numberof offspring produced daily once the aphid reached adulthood. Picturesof aphids were taken throughout the experiment to evaluate sizedifferences between treatment groups.

After 7 days of treatment, DNA was extracted from multiple aphids fromeach treatment group. Briefly, the aphid body surface was sterilized bydipping the aphid into a 6% bleach solution for approximately 5 seconds.Aphids were then rinsed in sterile water and DNA was extracted from eachindividual aphid using a DNA extraction kit (Qiagen, DNeasy kit)according to manufacturer's instructions. DNA concentration was measuredusing a nanodrop nucleic acid quantification, and Buchnera and aphid DNAcopy numbers were measured by qPCR. The primers used for Buchnera wereBuch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) andBuch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran,2016 PNAS). The primers used for aphid were ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

Antibiotic Treatment Delays and Stops Progression of Aphid Development

LSR-1 1^(st) instar aphids were divided into three separate treatmentgroups as defined in Experimental Design (above). Aphids were monitoreddaily and the number of aphids at each developmental stage wasdetermined. Aphids treated with artificial diet alone without essentialamino acids began reaching maturity (5th instar stage) at approximately6 days (FIG. 2A). Development was delayed in aphids treated withrifampicin, and by 6 days of treatment, most aphids did not maturefurther than the 3^(rd) instar stage, even after 12 days and their sizeis drastically affected (FIGS. 2A-2C).

In contrast, aphids treated with artificial diet with rifampicinsupplemented with essential amino acids developed faster and to higherinstar stages as compared to aphids treated with rifampicin alone, butnot as quickly as aphids treated with artificial diet without essentialamino acids (FIGS. 2A-2C). These data indicate that treatment withrifampicin impaired aphid development. Adding back essential amino acidspartially rescued this defect in development.

Antibiotic Treatment Increased Aphid Mortality

Survival rate of aphids was also measured during the treatments. Themajority of the aphids treated with artificial diet alone withoutessential amino acids were alive at 5 days post-treatment (FIG. 3).After 5 days, aphids began gradually dying, and some survived beyond 13days post-treatment.

In contrast, aphids treated with rifampicin without essential aminoacids had lower survival rates than aphids treated with artificial dietalone (p<0.00001). Rifampicin-treated aphids began dying after 1 day oftreatment and all aphids succumbed to treatment by 9 days. Aphidstreated with both rifampicin and essential amino acids survived longerthan those treated with rifampicin alone (p=0.017). These data indicatethat rifampicin treatment affected aphid survival.

Antibiotic Treatment Decreased Aphid Reproduction

Fecundity was also monitored in aphids during the treatments. By days 7and 8 post-treatment, the majority of the adult aphids treated withartificial diet without essential amino acids began reproducing. Themean number of offspring produced daily after maturity by aphids treatedwith artificial diet without essential amino acids was approximately 4(FIG. 4). In contrast, aphids treated with rifampicin with or withoutessential amino acids were unable to reach adulthood and produceoffspring. These data indicate that rifampicin treatment resulted in aloss of aphid reproduction.

Antibiotic Treatment Decreased Buchnera in Aphids

To test whether rifampicin, specifically resulted in loss of Buchnera inaphids, and that this loss impacted aphid fitness, DNA was extractedfrom aphids in each treatment group after 7 days of treatment and qPCRwas performed to determine the Buchnera/aphid copy numbers. Aphidstreated with artificial diet alone without essential amino acids hadhigh ratios of Buchnera/aphid DNA copies. In contrast, aphids treatedwith rifampicin had ˜4-fold less Buchnera/aphid DNA copies (FIG. 5),indicating that rifampicin treatment decreased Buchnera levels.

Leaf Coating Delivery Experimental Design

Rifampicin stock solution was added to 0.025% of a nonionicorganosilicone surfactant solvent, Silwet L-77, to obtain a finalconcentration of 50 μg/ml rifampicin. Aphids (eNASCO strain,Acyrthosiphon pisum) were grown on fava bean plants as described in aprevious Example. For experiments, first instar aphids were collectedfrom healthy plants and divided into 2 different treatment groups:leaves were sprayed with 1) negative control (solvent solution only), 2)50 μg/ml rifampicin in solvent. Solutions were absorbed onto a 2×2 cmpiece of fava bean leaf.

Each treatment group received approximately the same number ofindividuals from each of the collection plant. For each treatment, 20aphids were placed onto each leaf. Aphids were monitored daily forsurvival and dead aphids were removed when they were discovered. Inaddition, the developmental stage (1^(st), 2^(nd), 3^(rd), 4^(th),5^(th) instar, and 5R, representing a reproducing 5th instar) wasdetermined daily throughout the experiment. Pictures of aphids weretaken throughout the experiment to evaluate size differences betweentreatment groups.

After 6 days of treatment, DNA was extracted from multiple aphids fromeach treatment group and qPCR for quantifying Buchnera levels was doneas described in the previous Example.

Antibiotic Treatment Delivered Through Leaf Coating Delays and StopsProgression of Aphid Development

LSR-1 1^(st) instar aphids were divided into two separate treatmentgroups as defined in the Experimental Design described herein. Aphidswere monitored daily and the number of aphids at each developmentalstage was determined. Aphids placed on coated leaves treated withcontrol began reaching maturity (5^(th) instar reproducing stage; 5R) atapproximately 6 days (FIG. 6A). Development was delayed in aphids placedon coated leaves with rifampicin, and by 6 days of treatment, mostaphids did not mature further than the 3^(rd) instar stage, even after12 days, and their size is drastically affected (FIGS. 6A and 6B).

These data indicate that leaf coating with rifampicin impaired aphiddevelopment.

Antibiotic Treatment Delivered Through Leaf Coating Increased AphidMortality

Survival rate of aphids was also measured during the leaf coatingtreatments. Aphids placed on coated leaves with rifampicin had lowersurvival rates than aphids placed on coated leaves with the control(FIG. 7). These data indicate that rifampicin treatment deliveredthrough leaf coating affected aphid survival.

Antibiotic Treatment Delivered Through Leaf Coating Decreased Buchnerain Aphids

To test whether rifampicin delivered through leaf coating, specificallyresulted in loss of Buchnera in aphids, and that this loss impactedaphid fitness, DNA was extracted from aphids in each treatment groupafter 6 days of treatment and qPCR was performed to determine theBuchnera/aphid copy numbers.

Aphids placed on leaves treated with control had high ratios ofBuchnera/aphid DNA copies. In contrast, aphids placed on leaves treatedwith rifampicin had a drastic reduction of Buchnera/aphid DNA copies(FIG. 8), indicating that rifampicin leaf coating treatment eliminatedendosymbiotic Buchnera.

Microinjection Delivery Experimental Design:

Microinjection was performed using NanoJet III Auto-Nanoliter Injectorwith the in-house pulled borosilicate needle (Drummond Scientific;Cat#3-000-203-G/XL). Aphids (eNASCO strain, Acyrthosiphon pisum) weregrown on fava bean plants as described in a previous Example. Aphids aretransferred using a paint brush to a tubing system connected to vacuum(FIG. 1C). The injection site was at the ventral thorax of the aphid.The injection solutions were either the organosilicone surfactantsolvent 0.025% Silwet L-77 (Lehle Seeds, Cat No VIS-01) in water(negative control), or 50 μg/ml of rifampicin formulated in solventsolution. The injection volume was 10 nl for nymph and 20 nl for adult(both at a rate of 2 nl/sec). Each treatment group had approximately thesame number of individuals injected from each of the collection plants.After injection, aphids were released into a petri dish with fava beanleaves, whose stems are in 2% agar.

Microinjection with Antibiotic Treatment Decreased Buchnera in Aphids

To test whether rifampicin delivered by microinjection results in lossof Buchnera in aphids, and that this loss impacts aphid fitness asdemonstrated in previous Examples, DNA was extracted from aphids in eachtreatment group after 4 days of treatment and qPCR was performed asdescribed in a previous Example to determine the Buchnera/aphid copynumbers.

Aphids microinjected with negative control had high ratios ofBuchnera/aphid DNA copies. In contrast, aphid nymphs and adultsmicroinjected with rifampicin had a drastic reduction of Buchnera/aphidDNA copies (FIG. 9), indicating that rifampicin microinjection treatmentdecreased the presence of endosymbiotic Buchnera.

Topical Delivery Experimental Design:

Aphids (LSR-1 strain, Acyrthosiphon pisum) were grown on fava beanplants as described in a previous Example. Spray bottles were filledwith 2 ml of control (0.025% Silwet L-77) or rifampicin solutions (50μg/ml of in solvent solution). Aphids (number=10) were transferred tothe bottom of a clean petri dish and gathered to the corner of the petridish using a paint brush. Subsequently, aphids were separated into twocohorts and sprayed with ˜100 μl of either control or rifampicinsolutions. Immediately after spraying, the petri dish was covered with alid. Five minutes after spraying, aphids were released into a petri dishwith fava bean leaves with stems in 2% agar.

Topical Delivery of Antibiotic Treatment Decreased Buchnera in Aphids

To test whether rifampicin delivered by topical delivery results in lossof Buchnera in aphids, and that this loss impacts aphid fitness asdemonstrated in previous Examples, DNA was extracted from aphids in eachtreatment group after 3 days of treatment and qPCR as described in aprevious Example was performed to determine the Buchnera/aphid copynumbers.

Aphids sprayed with the control solution had high ratios ofBuchnera/aphid DNA copies. In contrast, aphids sprayed with rifampicinhad a drastic reduction of Buchnera/aphid DNA copies (FIG. 10),indicating that rifampicin treatment delivered through topical treatmentdecreases the presence of endosymbiotic Buchnera.

Leaf Injection Method A—Leaf Perfusion and Cutting

Experimental Design:

Aphids LSR-1 (which harbor only Buchnera), Acyrthosiphon pisum weregrown on fava bean plants (Vroma Vicia faba from Johnny's SelectedSeeds) in a climate-controlled incubator (16 h light/8 h darkphotoperiod; 60±5% RH; 25±2° C.). Prior to being used for aphid rearing,fava bean plants were grown in potting soil at 24° C. with 16 h of lightand 8 h of darkness. To limit maternal effects or health differencesbetween plants, 5-10 adults from different plants were distributed among10 two-week-old plants, and allowed to multiply to high density for 5-7days. For experiments, first and second instar aphids were collectedfrom healthy plants and divided into 2 different treatment groups: 1)negative control (leaf injected with water plus blue food coloring) and2) leaf injected with 50 μg/ml rifampicin in water plus blue foodcoloring. Each treatment group received approximately the same number ofindividuals from each of the collection plants. For treatment,rifampicin stock solution (25 mg/ml in 100% methanol) was diluted to 50μg/ml in water plus blue food coloring. The solution was then placedinto a 1.5 ml Eppendorf tube with a fava bean stem perfused with thesolutions and the opening of the tube was closed using parafilm. Thisfeeding system was then placed into a deep petri dish (FisherScientific, Cat# FB0875711) and aphids were applied to the leaves of theplant. For each treatment, 74-81 aphids were placed onto each leaf. Thefeeding systems were changed every 2-3 days throughout the experiment.Aphids were monitored daily for survival and dead aphids were removedfrom the deep petri dish when they were discovered. In addition, thedevelopmental stage (1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th) and 5R(5^(th) that has reproduced) instar) was determined daily throughout theexperiment.

Antibiotic Treatment Delivered Through Leaf Injection Method a Delaysand Stops Progression of Aphid Development

LSR-1 1st and 2nd instar aphids were divided into two separate treatmentgroups as defined in Leaf injection method A—Leaf perfusion and cuttingExperimental Design (described herein). Aphids were monitored daily andthe number of aphids at each developmental stage was determined. Aphidstreated with water plus food coloring began reaching maturity (5thinstar stage) at approximately 6 days (FIG. 11). Development was delayedin aphids feeding on rifampicin injected leaves, and by 6 days oftreatment, most aphids did not mature further than the 4th instar stage.Even after 8 days, the development of aphids feeding on rifampicininjected leaves was drastically delayed (FIG. 11). These data indicatethat rifampicin treatment via leaf perfusion impaired aphid development.

Antibiotic Treatment Delivered Through Leaf Injection Method A IncreasedAphid Mortality

Survival rate of aphids was also measured during the leaf perfusionexperiments. Aphids placed on leaves injected with rifampicin had lowersurvival rates than aphids placed on leaves injected with the controlsolution (FIG. 12). These data indicate that rifampicin treatmentdelivered through leaf injection affected aphid survival.

Antibiotic Treatment Delivered Thorough Leaf Injection Method a Resultsin Decreased Levels of Buchnera

To test whether rifampicin delivered via leaf perfusion results in lossof Buchnera in aphids, and that this loss impacts aphid fitness asdemonstrated in previous Examples, DNA was extracted from aphids in eachtreatment group after 8 days of treatment and qPCR as described in aprevious Example was performed to determine the Buchnera/aphid copynumbers.

Aphids feeding on leaves injected with the control solution had highratios of Buchnera/aphid DNA copies. In contrast, aphids feeding onleaves injected with rifampicin had a reduction of Buchnera/aphid DNAcopies (FIG. 13), indicating that rifampicin treatment delivered vialeaf injection decreases the presence of endosymbiotic Buchnera, asshown in previous Examples, and resulted in a fitness decrease.

Leaf Perfusion and Delivery Through Plant

Experimental Design:

Aphids LSR-1 (which harbor only Buchnera), Acyrthosiphon pisum weregrown on fava bean plants (Vroma Vicia faba from Johnny's SelectedSeeds) in a climate-controlled incubator (16 h light/8 h darkphotoperiod; 60±5% RH; 25±2° C.). Prior to being used for aphid rearing,fava bean plants were grown in potting soil at 24° C. with 16 h of lightand 8 h of darkness.

To limit maternal effects or health differences between plants, 5-10adults from different plants were distributed among 10 two-week-oldplants, and allowed to multiply to high density for 5-7 days. Forexperiments, first and second instar aphids were collected from healthyplants and divided into 2 different treatment groups: 1) aphids placedon leaves injected with the negative control solution (water and foodcoloring) and placed into an Eppendorf tube with the negative controlsolution, or 2) aphids placed on leaves that were injected with 100ug/ml rifampicin in water plus food coloring and put into an Eppendorftube with 100 ug/ml rifampicin in water. Each treatment group receivedapproximately the same number of individuals from each of the collectionplants.

For treatment, rifampicin stock solution (25 mg/ml in 100% methanol) wasdiluted to 100 μg/ml in water plus blue food coloring. The solution wasthen placed into a 1.5 ml Eppendorf tube with a fava bean stem with aleaf also perfused with the solutions and the opening of the tube wasclosed using parafilm. This feeding system was then placed into a deeppetri dish (Fisher Scientific, Cat# FB0875711) and aphids were appliedto the leaves of the plant.

For each treatment, 49-50 aphids were placed onto each leaf. The feedingsystems were changed every 2-3 days throughout the experiment. Aphidswere monitored daily for survival and dead aphids were removed from thedeep petri dish when they were discovered.

In addition, the developmental stage (1^(st), 2^(nd), 3^(rd), 4^(th),5^(th) and 5R (5^(th) that has reproduced) instar) was determined dailythroughout the experiment.

Antibiotic Treatment Delivered Through Leaf Injection and DeliveryThrough Plant Delays and Stops Progression of Aphid Development

LSR-1 1^(st) and 2^(nd) instar aphids were divided into two separatetreatment groups as defined in Leaf perfusion and delivery through plantExperimental Design (described herein). Aphids were monitored daily andthe number of aphids at each developmental stage was determined. Aphidstreated with the control solution (water plus food coloring only) beganreaching maturity (5^(th) instar stage) at approximately 6 days (FIG.14).

Development was delayed in aphids treated with rifampicin, and by 6 daysof treatment, most aphids did not mature further than the 3rd instarstage. Even after 8 days, their development was drastically delayed(FIG. 14). These data indicate that rifampicin treatment via leafperfusion impaired aphid development.

Antibiotic Treatment Delivered Through Leaf Injection and DeliveryThrough Plant Increased Aphid Mortality

Survival rate of aphids was also measured during the experiments whereaphids were treated with either control solution or rifampicin deliveredvia leaf perfusion and through the plant. Aphids feeding on leavesperfused and treated with rifampicin had lower survival rates thanaphids feeding on leaves perfused and treated with the control solution(FIG. 15). These data indicate that rifampicin treatment deliveredthrough leaf perfusion and through the plant negatively impacted aphidsurvival.

Antibiotic Treatment Delivered Via Leaf Injection and Through the PlantResults in Decreased Levels of Buchnera

To test whether rifampicin delivered via leaf perfusion and through theplant results in loss of Buchnera in aphids, and that this loss impactsaphid fitness as demonstrated in previous Examples, DNA was extractedfrom aphids in each treatment group after 6 and 8 days of treatment andqPCR was performed to determine the Buchnera/aphid copy numbers, asdescribed in previous Examples.

Aphids feeding on leaves injected and treated with the control solutionhad high ratios of Buchnera/aphid DNA copies. In contrast, aphidsfeeding on leaves perfused and treated with rifampicin had astatistically significant reduction of Buchnera/aphid DNA copies at bothtime points (p=0.0037 and p=0.0025 for days 6 and 8, respectively)(FIGS. 16A and 16B), indicating that rifampicin treatment delivered vialeaf perfusion and through the plant decreased the presence ofendosymbiotic Buchnera, and as shown in previous Examples, and resultedin a fitness decrease.

Combination Delivery Method

Experimental Design:

Aphids LSR-1 (which harbor only Buchnera), Acyrthosiphon pisum weregrown on fava bean plants (Vroma Vicia faba from Johnny's SelectedSeeds) in a climate-controlled incubator (16 h light/8 h darkphotoperiod; 60±5% RH; 20±2° C.). Prior to being used for aphid rearing,fava bean plants were grown in potting soil at 24° C. with 16 h of lightand 8 h of darkness. To limit maternal effects or health differencesbetween plants, 5-10 adults from different plants were distributed among10 two-week-old plants, and allowed to multiply to high density for 5-7days.

For experiments, first and second instar aphids were collected fromhealthy plants and divided into 2 different treatment groups: 1)treatment with Silwet-L77 or water control solutions or 2) treatmentwith rifampicin diluted in silwet L-77 or water. Each treatment groupreceived approximately the same number of individuals from each of thecollection plants. The combination of delivery methods was as follows:a) Topical delivery to aphid and plant by spraying 0.025% nonionicorganosilicone surfactant solvent Silwet L-77 (negative control) or 100μg/ml of rifampicin formulated in solvent solution using a 30 mL spraybottle and b) Delivery through plant with either water (negativecontrol) or 100 μg/ml of rifampicin formulated in water. For treatment,rifampicin stock solution (25 mg/ml in 100% methanol) was diluted to 100μg/ml in Silwet L-77 (for topical treatment to aphid and coating theleaf) or water (for delivery through plant). The solution was thenplaced into a 1.5 ml Eppendorf tube with a fava bean stem with a leafalso perfused with the solutions and the opening of the tube was closedusing parafilm. This feeding system was then placed into a deep petridish (Fisher Scientific, Cat# FB0875711) and aphids were applied to theleaves of the plant.

For each treatment, 76-80 aphids were placed onto each leaf. The feedingsystems were changed every 2-3 days throughout the experiment. Aphidswere monitored daily for survival and dead aphids were removed from thedeep petri dish when they were discovered.

In addition, the developmental stage (1^(st), 2^(nd), 3^(rd), 4^(th),5^(th), and 5R (5^(th) that has reproduced) instar) was determined dailythroughout the experiment.

Combination Antibiotic Treatment Delays Aphid Development

LSR-1 1^(st) and 2^(nd) instar aphids were divided into two separatetreatment groups as defined in Combination delivery method ExperimentalDesign (described herein). Aphids were monitored daily and the number ofaphids at each developmental stage was determined. Control treatedaphids began reaching maturity (5^(th) instar stage) at approximately 6days (FIG. 17). Development was delayed in aphids treated withrifampicin, and by 6 days of treatment, most aphids did not maturefurther than the 3^(rd) instar stage, even after 7 days theirdevelopment was drastically delayed (FIG. 17). These data indicate thata combination of rifampicin treatments impaired aphid development.

Combination Antibiotic Treatment Results in Increased Aphid Mortality

Survival rate of aphids was also measured during the experiments whereaphids were treated with a combination of rifampicin treatments.Rifampicin treated aphids had slightly lower survival rates than aphidstreated with control solutions (FIG. 18). These data indicate thatrifampicin treatment delivered through a combination of treatmentsaffected aphid survival.

Combination Antibiotic Treatment in Decreased Levels of Buchnera

To test whether rifampicin delivered via a combination of methodsresults in loss of Buchnera in aphids, and that this loss impacts aphidfitness as demonstrated in previous Examples, DNA was extracted fromaphids in each treatment group after 7 days of treatment and qPCR asdescribed in a previous Example was performed to determine theBuchnera/aphid copy numbers.

Aphids treated with the control solutions had high ratios ofBuchnera/aphid DNA copies. In contrast, aphids treated with rifampicinhad a statistically significant and drastic reduction of Buchnera/aphidDNA copies (p=0.227; FIG. 19), indicating that rifampicin treatmentdelivered via a combination of methods decreases the presence ofendosymbiotic Buchnera, and as shown in previous Examples, this resultedin a fitness decrease.

Together this data described in the previous Examples demonstrate theability to kill and decrease the development, reproductive ability,longevity, and endogenous bacterial populations, e.g., fitness, ofaphids by treating them with an antibiotic through multiple deliverymethods.

Example 15: Insects Treated with a Natural Antimicrobial Polysacharide

This Example demonstrates the treatment of aphids with Chitosan, anatural cationic linear polysaccharide of deacetylatedbeta-1,4-D-glucosamine derived from chitin. Chitin is the structuralelement in the exoskeleton of insects, crustaceans (mainly shrimp andcrabs) and cell walls of fungi, and the second most abundant naturalpolysaccharide after cellulose. This Example demonstrates that theeffect of chitosan on insects was mediated through the modulation ofbacterial populations endogenous to the insect that were sensitive tochitosan. One targeted bacterial strain is Buchnera aphidicola.

Therapeutic Design

The chitosan solution was formulated using a combination of leafperfusion and delivery through plants (FIG. 20). The control solutionwas leaf injected with water+blue food coloring and water in tube. Thetreatment solution with 300 ug/ml chitosan in water (low molecularweight) via leaf injection (with blue food coloring) and through plant(in Eppendorf tube).

Leaf Perfusion-Plant Delivery Experimental Design:

Aphids LSR-1 (which harbor only Buchnera), Acyrthosiphon pisum weregrown on fava bean plants (Vroma Vicia faba from Johnny's SelectedSeeds) in a climate-controlled incubator (16 h light/8 h darkphotoperiod; 60±5% RH; 25±2° C.). Prior to being used for aphid rearing,fava bean plants were grown in potting soil at 24° C. with 16 h of lightand 8 h of darkness. To limit maternal effects or health differencesbetween plants, 5-10 adults from different plants were distributed among10 two-week-old plants, and allowed to multiply to high density for 5-7days. For experiments, first and second instar aphids were collectedfrom healthy plants and divided into 2 different treatment groups: 1)negative control (water treated), 2) The treatment solution included 300ug/ml chitosan in water (low molecular weight). Each treatment groupreceived approximately the same number of individuals from each of thecollection plants.

Chitosan (Sigma, catalog number 448869-50G) stock solution was made at1% in acetic acid, sterilized autoclaving, and stored at 4° C. Fortreatment (see Therapeutic design), the appropriate amount of stocksolution was diluted with water to obtain the final treatmentconcentration of chitosan. The solution was then placed into a 1.5 mlEppendorf tube with a fava bean stem with a leaf also perfused with thesolutions and the opening of the tube was closed using parafilm. Thisfeeding system was then placed into a deep petri dish (FisherScientific, Cat# FB0875711) and aphids were applied to the leaves of theplant.

For each treatment, 50-51 aphids were placed onto each leaf. The feedingsystems were changed every 2-3 days throughout the experiment. Aphidswere monitored daily for survival and dead aphids were removed from thedeep petri dish when they were discovered.

In addition, the developmental stage (1^(st), 2^(nd), 3^(rd), 4^(th),5^(th), and 5R (5^(th) that has reproduced) instar) was determined dailythroughout the experiment.

After 8 days of treatment, DNA was extracted from multiple aphids fromeach treatment group. Briefly, the aphid body surface was sterilized bydipping the aphid into a 6% bleach solution for approximately 5 seconds.Aphids were then rinsed in sterile water and DNA was extracted from eachindividual aphid using a DNA extraction kit (Qiagen, DNeasy kit)according to manufacturer's instructions. DNA concentration was measuredusing a nanodrop nucleic acid quantification, and Buchnera and aphid DNAcopy numbers were measured by qPCR. The primers used for Buchnera wereBuch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) andBuch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran,2016 PNAS). The primers used for aphid were ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

There was a Negative Response on Insect Fitness Upon Treatment with theNatural Antimicrobial Polysaccharide

LSR-1 A. pisum 1^(st) and 2^(nd) instar aphids were divided into twoseparate treatment groups as defined in Experimental Design (above).Aphids were monitored daily and the number of aphids at eachdevelopmental stage was determined. Aphids treated with the negativecontrol alone began reaching maturity (5th instar stage) atapproximately 6 days (FIG. 21). Development was delayed in aphidstreated with chitosan solution, and by 6 days of treatment withchitosan, most aphids did not mature further than the 4^(rd) instarstage. These data indicate that treatment with chitosan delayed andstopped progression of aphid development.

Chitosan Treatment Increased Aphid Mortality

Survival rate of aphids was also measured during the treatments. Themajority of the aphids treated with the control alone were alive at 3days post-treatment (FIG. 22). After 4 days, aphids began graduallydying, and some survived beyond 7 days post-treatment.

In contrast, aphids treated with chitosan solution had lower survivalrates than aphids treated with control. These data indicate that therewas a decrease in survival upon treatment with the natural antimicrobialpolysaccharide.

Chitosan Treatment Decreased Buchnera in Aphids

To test whether the chitosan solution treatment, specifically resultedin loss of Buchnera in aphids, and that this loss impacted aphidfitness, DNA was extracted from aphids in each treatment group after 8days of treatment and qPCR was performed to determine the Buchnera/aphidcopy numbers. Aphids treated with control alone had high ratios ofBuchnera/aphid DNA copies. In contrast, aphids treated with 300 ug/mlchitosan in water had ˜5-fold less Buchnera/aphid DNA copies (FIG. 23),indicating that chitosan treatment decreased Buchnera levels.

Together this data described previously demonstrated the ability to killand decrease the development, longevity, and endogenous bacterialpopulations, e.g., fitness, of aphids by treating them with a naturalantimicrobial polysaccharide.

Example 16: Insects Treated with Nisin, a Natural Antimicrobial Peptide

This Example demonstrates the treatment of aphids with the natural,“broad spectrum,” polycyclic antibacterial peptide produced by thebacterium Lactococcus lactis that is commonly used as a foodpreservative. The antibacterial activity of nisin is mediated throughits ability to generate pores in the bacterial cell membrane andinterrupt bacterial cell-wall biosynthesis through a specific lipid IIinteraction. This Example demonstrates that the negative effect of nisinon insect fitness is mediated through the modulation of bacterialpopulations endogenous to the insect that were sensitive to nisin. Onetargeted bacterial strain is Buchnera aphidicola.

Therapeutic Design:

Nisin was formulated using a combination of leaf perfusion and deliverythrough plants. The control solution (water) or treatment solution(nisin) was injected into the leaf and placed in the Eppendorf tube. Thetreatment solutions consisted of 1.6 or 7 mg/ml nisin in water.

Leaf perfusion-Plant Delivery Experimental Design:

LSR-1 aphids, Acyrthosiphon pisum were grown on fava bean plants (VromaVicia faba from Johnny's Selected Seeds) in a climate-controlledincubator (16 h light/8 h dark photoperiod; 60±5% RH; 25±2° C.). Priorto being used for aphid rearing, fava bean plants were grown in pottingsoil at 24° C. with 16 h of light and 8 h of darkness. To limit maternaleffects or health differences between plants, 5-10 adults from differentplants were distributed among 10 two-week-old plants, and allowed tomultiply to high density for 5-7 days. For experiments, first and secondinstar aphids were collected from healthy plants and divided into 2different treatment groups: 1) negative control (water treated), 2)nisin treated with either 1.6 or 7 mg/ml nisin in water. Each treatmentgroup received approximately the same number of individuals from each ofthe collection plants.

For treatment (see Therapeutic design), nisin (Sigma, product number:N5764) solution was made at 1.6 or 7 mg/ml (w/v) in water and filtersterilized using a 0.22 um syringe filter. The solution was theninjected into the leaf of the plant and the stem of the plant was placedinto a 1.5 ml Eppendorf tube. The opening of the tube was closed usingparafilm. This feeding system was then placed into a deep petri dish(Fisher Scientific, Cat# FB0875711) and aphids were applied to theleaves of the plant.

For each treatment, 56-59 aphids were placed onto each leaf. The feedingsystems were changed every 2-3 days throughout the experiment. Aphidswere monitored daily for survival and dead aphids were removed from thedeep petri dish when they were discovered.

In addition, the developmental stage (1st, 2nd, 3rd, 4th, 5th, and 5R(5th instar aphids that are reproducing) instar) was determined dailythroughout the experiment.

After 8 days of treatment, DNA was extracted from the remaining aphidsin each treatment group. Briefly, the aphid body surface was sterilizedby dipping the aphid into a 6% bleach solution for approximately 5seconds. Aphids were then rinsed in sterile water and DNA was extractedfrom each individual aphid using a DNA extraction kit (Qiagen, DNeasykit) according to manufacturer's instructions. DNA concentration wasmeasured using a nanodrop nucleic acid quantification, and Buchnera andaphid DNA copy numbers were measured by qPCR. The primers used forBuchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) andBuch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran,2016 PNAS). The primers used for aphid were ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

There was a Dose-Dependent Negative Response on Insect Fitness UponTreatment with Nisin

LSR-1 A. pisum 1st and 2nd instar aphids were divided into threeseparate treatment groups as defined in Experimental Design (above).Aphids were monitored daily and the number of aphids at eachdevelopmental stage was determined. Aphids treated with the negativecontrol solution (water) began reaching maturity (5th instar stage) atapproximately 6 days, and reproducing (5R stage) by 7 days (FIG. 24).Development was severely delayed in aphids treated with 7 mg/ml nisin.Aphids treated with 7 mg/ml nisin only developed to the 2nd instar stageby day 3, and by day 6, all aphids in the group were dead (FIG. 24).Development was slightly delayed in aphids treated with the lowerconcentration of nisin (1.6 mg/ml) and at each time point assessed,there were more less-developed aphids compared to water-treated controls(FIG. 24). These data indicate that treatment with nisin delayed andstopped progression of aphid development and this delay in developmentwas dependent on the dose of nisin administered.

However, it is important to note that treatment with 7 mg/ml of nisinalso had a negative impact on the health of the leaves used in theassay. Shortly after leaf perfusion of 7 mg/ml of nisin it startedturning brown. After two days, the leaves perfused with 7 mg/ml turnedblack. There was no noticeable difference in the condition of the leavestreated with 1.6 mg/ml nisin.

Treatment with Nisin Resulted in Increased Aphid Mortality

Survival rate of aphids was also measured during the treatments.Approximately 50% of aphids treated with the control alone survived the8-day experiment (FIG. 25). In contrast, survival was significantly lessfor aphids treated with 7 mg/ml nisin (p<0.0001, by Log-Rank Mantel Coxtest), and all aphids in this group succumbed to the treatment by 6 days(FIG. 25). Aphids treated with the lower dose of nisin (1.6 mg/ml) hadhigher mortality compared to control treated aphids, although thedifference did not reach statistical significance (p=0.0501 by Log-RankMantel Cox test). These data indicate that there was a dose-dependentdecrease in survival upon treatment with nisin.

Treatment with Nisin Resulted in Decreased Buchnera in Aphids

To test whether treatment with nisin, specifically resulted in loss ofBuchnera in aphids, and that this loss impacted aphid fitness, DNA wasextracted from aphids in each treatment group after 8 days of treatmentand qPCR was performed to determine the Buchnera/aphid copy numbers.Aphids treated with control alone had high ratios of Buchnera/aphid DNAcopies. In contrast, aphids treated with 1.6 mg/ml nisin had ˜1.4-foldless Buchnera/aphid DNA copies after 8 days of treatment (FIG. 26). Noaphids were alive in the group treated with 7 mg/ml nisin, andtherefore, Buchnera/aphid DNA copies was not assessed in this group.These data indicate that nisin treatment decreased Buchnera levels.

Together this data described previously demonstrate the ability to killand decrease the development, longevity, and endogenous bacterialpopulations, e.g., fitness, of aphids by treating them with theantimicrobial peptide nisin.

Example 17: Insects Treated with Levulinic Acid Decreases Insect Fitness

This Example demonstrates the treatment of aphids with levulinic acid, aketo acid produced by heating a carbohydrate with hexose (e.g., wood,starch, wheat, straw, or cane sugar) with the addition of a dilutemineral acid reduces insect fitness. Levulinic acid has previously beentested as an antimicrobial agent against Escherichia coli and Salmonellain meat production (Carpenter et al., 2010, Meat Science; Savannah G.Hawkins, 2014, University of Tennessee Honors Thesis). This Exampledemonstrates that the effect of levulinic acid on insects was mediatedthrough the modulation of bacterial populations endogenous to the insectthat were sensitive to levulinic acid. One targeted bacterial strain isBuchnera aphidicola.

Therapeutic Design:

The levulinic acid was formulated using a combination of leaf perfusionand delivery through plants. The control solution was leaf injected withwater and water was placed in the Eppendorf tube. The treatmentsolutions included 0.03 or 0.3% levulinic acid in water via leafinjection and through plant (in Eppendorf tube).

Leaf Perfusion-Plant Delivery Experimental Design:

eNASCO aphids, Acyrthosiphon pisum were grown on fava bean plants (VromaVicia faba from Johnny's Selected Seeds) in a climate-controlledincubator (16 h light/8 h dark photoperiod; 60±5% RH; 25±2° C.). Priorto being used for aphid rearing, fava bean plants were grown in pottingsoil at 24° C. with 16 h of light and 8 h of darkness. To limit maternaleffects or health differences between plants, 5-10 adults from differentplants were distributed among 10 two-week-old plants, and allowed tomultiply to high density for 5-7 days. For experiments, first and secondinstar aphids were collected from healthy plants and divided into 2different treatment groups: 1) negative control (water treated), 2) Thetreatment solution included 0.03 or 0.3% levulinic acid in water. Eachtreatment group received approximately the same number of individualsfrom each of the collection plants.

For treatment (see Therapeutic design), levulinic acid (Sigma, productnumber: W262706) was diluted to either 0.03 or 0.3% in water. Thesolution was then placed into a 1.5 ml Eppendorf tube with a fava beanstem with a leaf also perfused with the solutions and the opening of thetube was closed using parafilm. This feeding system was then placed intoa deep petri dish (Fisher Scientific, Cat# FB0875711) and aphids wereapplied to the leaves of the plant.

For each treatment, 57-59 aphids were placed onto each leaf. The feedingsystems were changed every 2-3 days throughout the experiment. Aphidswere monitored daily for survival and dead aphids were removed from thedeep petri dish when they were discovered.

In addition, the developmental stage (1^(st), 2^(nd), 3^(rd), 4^(th),and 5^(th) instar) was determined daily throughout the experiment.

After 7 of treatment, DNA was extracted from the remaining aphids ineach treatment group. Briefly, the aphid body surface was sterilized bydipping the aphid into a 6% bleach solution for approximately 5 seconds.Aphids were then rinsed in sterile water and DNA was extracted from eachindividual aphid using a DNA extraction kit (Qiagen, DNeasy kit)according to manufacturer's instructions. DNA concentration was measuredusing a nanodrop nucleic acid quantification, and Buchnera and aphid DNAcopy numbers were measured by qPCR. The primers used for Buchnera wereBuch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) andBuch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran,2016 PNAS). The primers used for aphid were ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

There was a Dose-Dependent Negative Response on Insect Fitness UponTreatment with Levulinic Acid

eNASCO A. pisum 1^(st) and 2^(nd) instar aphids were divided into threeseparate treatment groups as defined in Experimental Design (above).Aphids were monitored daily and the number of aphids at eachdevelopmental stage was determined. Aphids treated with the negativecontrol alone began reaching maturity (5^(th) instar stage) atapproximately 7 days (FIG. 27). Development was delayed in aphidstreated with levulinic acid and by 11 days post-treatment, nearly allcontrol treated aphids reached maturity while ˜23 and 63% aphids treatedwith 0.03 and 0.3% levulinic acid, respectively, did not mature furtherthan the 4^(rd) instar stage. These data indicate that treatment withlevulinic acid delayed and stopped progression of aphid development andthis delay in development is dependent on the dose of levulinic acidadministered.

Treatment with Levulinic Acid Results in Increased Aphid Mortality

Survival rate of aphids was also measured during the treatments.Approximately 50% of aphids treated with the control alone survived the11-day experiment (FIG. 28). In contrast, survival was significantlyless for aphids treated with 0.3% levulinic acid (p<0.01). Aphidstreated with the low dose of levulinic acid (0.03%) had higher mortalitycompared to control treated aphids, although the difference did notreach statistical significance. These data indicate that there was adose-dependent decrease in survival upon treatment with levulinic acid.

Treatment with Levulinic Acid Results in Decreased Buchnera in Aphids

To test whether treatment with levulinic acid, specifically resulted inloss of Buchnera in aphids, and that this loss impacted aphid fitness,DNA was extracted from aphids in each treatment group after 7 days oftreatment and qPCR was performed to determine the Buchnera/aphid copynumbers. Aphids treated with control alone had high ratios ofBuchnera/aphid DNA copies. In contrast, aphids treated with 0.03 or 0.3%levulinic acid in water had ˜6-fold less Buchnera/aphid DNA copies after7 days of treatment (FIG. 29, left panel). These data indicate thatlevulinic acid treatment decreased Buchnera levels.

Together this data described previously demonstrated the ability to killand decrease the development, longevity, and endogenous bacterialpopulations, e.g., fitness, of aphids by treating them with levulinicacid.

Example 18: Insects Treated with a Plant Derived Secondary MetaboliteSolution

This Example demonstrates the treatment of aphids with gossypol aceticacid, a natural phenol derived from the cotton plant (genus Gossypium)that permeates cells and acts as an inhibitor for several dehydrogenaseenzymes. This Example demonstrates that the effect of gossypol oninsects was mediated through the modulation of bacterial populationsendogenous to the insect that were sensitive to gossypol. One targetedbacterial strain is Buchnera aphidicola.

Therapeutic Design:

The gossypol solution was formulated depending on the delivery method:

1) Through the plants: with 0 (negative control) or 0.5%, 0.25%, and0.05% of gossypol formulated in an artificial diet (based on Akey andBeck, 1971; see Experimental Design) without essential amino acids(histidine, isoleucine, leucine, lysine, methionine, phenylalanine,threonine, tryptophan, and valine).

2) Microinjection: injection solutions were either 0.5% of gossypol orartificial diet only (negative control).

Plant Delivery Experimental Design:

Aphids (either eNASCO (which harbor both Buchnera and Serratia primaryand secondary symbionts, respectively) or LSR-1 (which harbor onlyBuchnera) strains, Acyrthosiphon pisum) were grown on fava bean plants(Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlledincubator (16 h light/8 h dark photoperiod; 60±5% RH; 25±2° C.). Priorto being used for aphid rearing, fava bean plants were grown in pottingsoil at 24° C. with 16 h of light and 8 h of darkness. To limit maternaleffects or health differences between plants, 5-10 adults from differentplants were distributed among 10 two-week-old plants, and allowed tomultiply to high density for 5-7 days. For experiments, first and secondinstar aphids were collected from healthy plants and divided into 4different treatment groups: 1) artificial diet alone without essentialamino acids, 2) artificial diet alone without essential amino acids and0.05% of gossypol, 3) artificial diet alone without essential aminoacids and 0.25% of gossypol, and 4) artificial diet alone withoutessential amino acids and 0.5% of gossypol. Each treatment groupreceived approximately the same number of individuals from each of thecollection plants.

The artificial diet used was made as previously published (Akey andBeck, 1971 Continuous Rearing of the Pea Aphid, Acyrthosiphon pisum, ona Holidic Diet), with and without the essential amino acids (2 mg/mlhistidine, 2 mg/ml isoleucine, 2 mg/ml leucine, 2 mg/ml lysine, 1 mg/mlmethionine, 1.62 mg/ml phenylalanine, 2 mg/ml threonine, 1 mg/mltryptophan, and 2 mg/ml valine), except neither diet included homoserineor beta-alanyltyrosine. The pH of the diets was adjusted to 7.5 with KOHand diets were filter sterilized through a 0.22 μm filter and stored at4° C. for short term (<7 days) or at −80° C. for long term.

Gossypol acetic acid (Sigma, Cat#G4382-250MG) stock solution was made at5% in methanol and sterilized by passing through a 0.22 μm syringefilter, and stored at 4° C. For treatments (see Therapeutic design), theappropriate amount of stock solution was added to the artificial diet toobtain the different final concentrations of gossypol. The diet was thenplaced into a 1.5 ml Eppendorf tube with a fava bean stem with a leafand the opening of the tube was closed using parafilm. This feedingsystem was then placed into a deep petri dish (Fisher Scientific, Cat#FB0875711) and aphids were applied to the leaves of the plant.

For each treatment, 15-87 aphids were placed onto each leaf. Artificialdiet feeding systems were changed every 2-3 days throughout theexperiment. Aphids were monitored daily for survival and dead aphidswere removed from the deep petri dish housing the artificial feedingsystem when they were discovered.

In addition, the developmental stage (1^(st), 2^(nd), 3^(rd), 4^(th),5^(th), and 5R (5^(th) that has reproduced) instar) was determined dailythroughout the experiment. Once an aphid reached the 4th instar stage,they were given their own artificial feeding system in a deep petri dishso that fecundity could be monitored once they reached adulthood.

For adult aphids, new nymphs were counted daily and then discarded. Atthe end of the experiments, fecundity was measured in two ways: 1) themean day at which the first offspring for the treatment group wasdetermined and 2) the mean number of offspring produced daily once theaphid reached adulthood. Pictures of aphids were taken throughout theexperiment to evaluate size differences between treatment groups.

After 5 or 13 days of treatment, DNA was extracted from multiple aphidsfrom each treatment group. Briefly, the aphid body surface wassterilized by dipping the aphid into a 6% bleach solution forapproximately 5 seconds. Aphids were then rinsed in sterile water andDNA was extracted from each individual aphid using a DNA extraction kit(Qiagen, DNeasy kit) according to manufacturer's instructions. DNAconcentration was measured using a nanodrop nucleic acid quantification,and Buchnera and aphid DNA copy numbers were measured by qPCR. Theprimers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG;SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO:239) (Chong and Moran, 2016 PNAS). The primers used for aphid wereApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

There was a Dose-Dependent Negative Response on Insect Fitness UponTreatment with the Allelochemical Gossypol

eNASCO and LSR-1 A. pisum 1^(st) and 2^(nd) instar aphids were dividedinto four separate treatment groups as defined in Experimental Design(described herein). Aphids were monitored daily and the number of aphidsat each developmental stage was determined. Aphids treated withartificial diet alone began reaching maturity (5^(th) instar stage) atapproximately 3 days (FIG. 30A). Development was delayed in aphidstreated with gossypol, and by 5 days of treatment with 0.5% of gossypol,most aphids did not mature further than the 3^(rd) instar stage, andtheir size is also affected (FIGS. 30A and 30B). These data indicatethat treatment with gossypol delayed and stopped progression of aphiddevelopment, and that this response was dose dependent.

Gossypol Treatment Increased Aphid Mortality

Survival rate of aphids was also measured during the treatments. Themajority of the aphids treated with artificial diet alone withoutessential amino acids were alive at 2 days post-treatment (FIG. 31).After 4 days, aphids began gradually dying, and some survived beyond 7days post-treatment.

In contrast, aphids treated with 0.25 (not significantly different thancontrol, p=0.2794) and 0.5% of gossypol had lower survival rates thanaphids treated with artificial diet alone, with 0.5% gossypol treatmentbeing significantly different than AD no EAA control (p=0.002). 0.5%gossypol-treated aphids began dying after 2 days of treatment and allaphids succumbed to treatment by 4 days. Aphids treated with 0.25%survived a bit longer than those treated with 0.5% but were alsodrastically affected. These data indicate that there was adose-dependent decrease in survival upon treatment with theallelochemical gossypol.

Gossypol Treatment Decreased Aphid Reproduction

Fecundity was also monitored in aphids during the treatments. By days 7and 8 post-treatment, the majority of the adult aphids treated withartificial diet without essential amino acids began reproducing. Themean number of offspring produced daily after maturity by aphids treatedwith artificial diet without essential amino acids was approximately 5(FIGS. 32A and 32B).

In contrast, aphids treated with 0.25% of gossypol show a reduction toreach adulthood and produce offspring. These data indicate that gossypoltreatment resulted in a decrease of aphid reproduction.

Gossypol Treatment Decreased Buchnera in Aphids

To test whether different concentrations of gossypol, specificallyresulted in loss of Buchnera in aphids, and that this loss impactedaphid fitness, DNA was extracted from aphids in each treatment groupafter 5 or 13 days of treatment and qPCR was performed to determine theBuchnera/aphid copy numbers. Aphids treated with artificial diet alonewithout essential amino acids (control) had high ratios ofBuchnera/aphid DNA copies. In contrast, aphids treated with 0.25 and0.5% of gossypol had ˜4-fold less Buchnera/aphid DNA copies (FIG. 33),indicating that gossypol treatment decreased Buchnera levels, and thatthis decrease was concentration dependent.

Microinjection Delivery Experimental Design:

Microinjection was performed using NanoJet III Auto-Nanoliter Injectorwith the in-house pulled borosilicate needle (Drummond Scientific;Cat#3-000-203-G/XL). Aphids (LSR-1 strain, A. pisum) were grown on favabean plants as described in a previous Example. Each treatment group hadapproximately the same number of individuals injected from each of thecollection plants. Nymph aphids (<3^(rd) instar stage) were transferredusing a paint brush to a tubing system connected to vacuum andmicroinjected into the ventral thorax with 20 nl of either artificialdiet without essential amino acids (negative control) or 0.05% ofgossypol solution in artificial diet without essential amino acids.After injection, aphids were placed in a deep petri dish with a favabean leaf with stem in 2% agar.

Microinjection with Antibiotic Treatment Decreased Buchnera in Aphids

To test whether gossypol delivered by microinjection results in loss ofBuchnera in aphids, and that this loss impacts aphid fitness asdemonstrated in previous Examples, DNA was extracted from aphids in eachtreatment group after 4 days of treatment and qPCR was performed asdescribed in a previous Example to determine the Buchnera/aphid copynumbers.

Aphids microinjected with negative control had high ratios ofBuchnera/aphid DNA copies. In contrast, aphid nymphs and adultsmicroinjected with gossypol had a drastic reduction of Buchnera/aphidDNA copies (FIG. 34), indicating that gossypol microinjection treatmentdecreases the presence of endosymbiotic Buchnera, and as shown inprevious Examples this resulted in a fitness decrease.

Together this data described in the previous Examples demonstrated theability to kill and decrease the development, reproductive ability,longevity, and endogenous bacterial populations, e.g., fitness, ofaphids by treating them with plant secondary metabolite solution throughmultiple delivery methods.

Example 19: Insects Treated with Natural Plant Derived AntimicrobialCompound, Trans-Cinnemaldehyde

This Example demonstrates the treatment of aphids withtrans-cinnemaldehyde, a natural aromatic aldehyde that is the majorcomponent of bark extract of cinnamon (Cinnamomum zeylandicum) resultsin decreased fitness. Trans-cinnemaldehyde has been shown to haveantimicrobial activity against both gram-negative and gram-positiveorganisms, although the exact mechanism of action of its antimicrobialactivity remains unclear. Trans-cinnemaldehyde may damage bacterial cellmembranes and inhibit of specific cellular processes or enzymes (Gilland Holley, 2004 Applied Environmental Microbiology). This Exampledemonstrates that the effect of trans-cinnemaldehyde on insects wasmediated through the modulation of bacterial populations endogenous tothe insect that were sensitive to trans-cinnemaldehyde. One targetedbacterial strain is Buchnera aphidicola.

Therapeutic Design:

Trans-cinnemaldehyde was diluted to 0.05%, 0.5%, or 5% in water and wasdelivered through leaf perfusion (˜1 ml was injected into leaves) andthrough plants.

Experimental Design:

Aphids (LSR-1 (which harbor only Buchnera) strains, Acyrthosiphon pisum)were grown on fava bean plants (Vroma Vicia faba from Johnny's SelectedSeeds) in a climate-controlled incubator (16 h light/8 h darkphotoperiod; 60±5% RH; 25±2° C.). Prior to being used for aphid rearing,fava bean plants were grown in potting soil at 24° C. with 16 h of lightand 8 h of darkness. To limit maternal effects or health differencesbetween plants, 5-10 adults from different plants were distributed among10 two-week-old plants, and allowed to multiply to high density for 5-7days. For experiments, first and second instar aphids were collectedfrom healthy plants and divided into four different treatment groups: 1)water treated controls, 2) 0.05% trans-cinnemaldehyde in water, 3) 0.5%trans-cinnemaldehyde in water, and 4) 5% trans-cinnemaldehyde in water.Each treatment group received approximately the same number ofindividuals from each of the collection plants.

Trans-cinnemaldehyde (Sigma, Cat#C80687) was diluted to the appropriateconcentration in water (see Therapeutic design), sterilized by passingthrough a 0.22 μm syringe filter, and stored at 4° C. Fava bean leaveswere injected with approximately 1 ml of the treatment and then the leafwas placed in a 1.5 ml Eppendorf tube containing the same treatmentsolution. The opening of the tube where the fava bean stem was placedwas closed using parafilm. This treatment feeding system was then placedinto a deep petri dish (Fisher Scientific, Cat# FB0875711) and aphidswere applied to the leaves of the plant.

For each treatment, 40-49 aphids were placed onto each leaf. Treatmentfeeding systems were changed every 2-3 days throughout the experiment.Aphids were monitored daily for survival and dead aphids were removedfrom the deep petri dish housing the treatment feeding system when theywere discovered.

In addition, the developmental stage (1^(st), 2^(nd), 3^(rd), 4^(th),5^(th), and 5R (5^(th) that has reproduced) instar) was determined dailythroughout the experiment.

After 3 days of treatment, DNA was extracted from the remaining livingaphids from each treatment group. Briefly, the aphid body surface wassterilized by dipping the aphid into a 6% bleach solution forapproximately 5 seconds. Aphids were then rinsed in sterile water andDNA was extracted from each individual aphid using a DNA extraction kit(Qiagen, DNeasy kit) according to manufacturer's instructions. DNAconcentration was measured using a nanodrop nucleic acid quantification,and Buchnera and aphid DNA copy numbers were measured by qPCR. Theprimers used for Buchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG;SEQ ID NO: 238) and Buch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO:239) (Chong and Moran, 2016 PNAS). The primers used for aphid wereApEF1a 107F (CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

There was a Dose-Dependent Negative Response on Insect Fitness UponTreatment with the Natural Antimicrobial Trans-Cinnemaldehyde

LSR-1 A. pisum 1^(st) and 2^(nd) instar aphids were divided into fourseparate treatment groups as defined in Experimental Design (describedherein). Aphids were monitored daily and the number of aphids at eachdevelopmental stage was determined. Aphids treated with water alonebegan reaching the 3^(rd) instar stage at 3 days post-treatment (FIG.35). Development was delayed in aphids treated withtrans-cinnemaldehyde, and by 3 days of treatment with each the three ofthe trans-cinnemaldehyde concentrations, none of the aphids matured pastthe second instar stage (FIG. 35). Moreover, all the aphids treated withthe highest concentration of trans-cinnemaldehyde (5%) remained at the1^(st) instar stage throughout the course of the experiment. These dataindicate that treatment with trans-cinnemaldehyde delays and stopsprogression of aphid development, and that this response is dosedependent.

Trans-Cinnemaldehyde Treatment Increased Aphid Mortality

Survival rate of aphids was also measured during the treatments.Approximately 75 percent of the aphids treated with water alone werealive at 3 days post-treatment (FIG. 36). In contrast, aphids treatedwith 0.05%, 0.5%, and 5% trans-cinnemaldehyde had significantly lower(p<0.0001 for each treatment group compared to water treated control)survival rates than aphids treated with water alone. These data indicatethat there was a dose-dependent decrease in survival upon treatment withthe natural antimicrobial trans-cinnemaldehyde.

Trans-Cinnemaldehyde Treatment Decreased Buchnera in Aphids

To test whether different concentrations of trans-cinnemaldehyde,specifically resulted in loss of Buchnera in aphids, and that this lossimpacted aphid fitness, DNA was extracted from aphids in each treatmentgroup after 3 days of treatment and qPCR was performed to determine theBuchnera/aphid copy numbers. Aphids treated with water alone (control)had high ratios of Buchnera/aphid DNA copies. Similarly, aphids treatedwith the lowest concentration of trans-cinnemaldehyde (0.5%) had highratios of Buchnera/aphid DNA copies.

In contrast, aphids treated with 0.5 and 5% of trans-cinnemaldehyde had˜870-fold less Buchnera/aphid DNA copies (FIG. 37), indicating thattrans-cinnemaldehyde treatment decreased Buchnera levels, and that thisdecrease was concentration dependent.

Together this data described in the previous Examples demonstrate theability to kill and decrease the development, reproductive ability,longevity, and endogenous bacterial populations, e.g., fitness, ofaphids by treating them with plant secondary metabolite solution throughmultiple delivery methods.

Example 20: Insects Treated with Scorpion Antimicrobial Peptides

This Example demonstrates the treatment of aphids with multiple scorpionantimicrobial peptides (AMP), of which several are identified AMPs inthe venom gland transcriptome of the scorpion Urodacus yaschenkoi(Luna-Ramirez et al., 2017, Toxins). AMPs typically have a net positivecharge and hence, a high affinity for bacterial membranes. This Exampledemonstrates that the effect of the AMP on insects was mediated throughthe modulation of bacterial populations endogenous to the insect thatwere sensitive to AMP peptides. One targeted bacterial strain isBuchnera aphidicola, an obligate symbiont of aphids.

Therapeutic Design:

The Uy192 solution was formulated using a combination of leaf perfusionand delivery through plants. The control solution was leaf injected withwater+blue food coloring and water in tube. The treatment solutionconsisted of 100 ug/ml Uy192 in water via leaf injection (with blue foodcoloring) and through plant (in Eppendorf tube).

Leaf Perfusion-Plant Delivery Experimental Design:

Aphids LSR-1 (which harbor only Buchnera), Acyrthosiphon pisum weregrown on fava bean plants (Vroma Vicia faba from Johnny's SelectedSeeds) in a climate-controlled incubator (16 h light/8 h darkphotoperiod; 60±5% RH; 20±2° C.). Prior to being used for aphid rearing,fava bean plants were grown in potting soil at 24° C. with 16 h of lightand 8 h of darkness. To limit maternal effects or health differencesbetween plants, 5-10 adults from different plants were distributed among10 two-week-old plants, and allowed to multiply to high density for 5-7days. For experiments, first and second instar aphids were collectedfrom healthy plants and divided into 2 different treatment groups: 1)negative control (water treated), 2) The treatment solution of 100 ug/mlAMP in water. Each treatment group received approximately the samenumber of individuals from each of the collection plants.

Uy192 was synthesized by Bio-Synthesis at >75% purity. 1 mg oflyophilized peptide was reconstituted in 500 ul of 80% acetonitrile, 20%water, and 0.1% TFA, 100 ul (100 ug) was aliquoted into 10 individualEppendorf tubes, and allowed to dry. For treatment (see Therapeuticdesign), 1 ml of water was added to a 100 ug aliquot of peptide toobtain the final concentration of Uy192 (100 ug/ml). The solution wasthen placed into a 1.5 ml Eppendorf tube with a fava bean stem with aleaf also perfused with the solutions and the opening of the tube wasclosed using parafilm. This feeding system was then placed into a deeppetri dish (Fisher Scientific, Cat# FB0875711) and aphids were appliedto the leaves of the plant.

For each treatment, 50 aphids were placed onto each leaf. The feedingsystems were changed every 2-3 days throughout the experiment. Aphidswere monitored daily for survival and dead aphids were removed from thedeep petri dish when they were discovered.

In addition, the developmental stage (1^(st), 2^(nd), 3^(rd), 4^(th),5^(th), and 5R (5^(th) that has reproduced) instar) was determined dailythroughout the experiment.

After 8 days of treatment, DNA was extracted from the remaining aphidsin each treatment group. Briefly, the aphid body surface was sterilizedby dipping the aphid into a 6% bleach solution for approximately 5seconds. Aphids were then rinsed in sterile water and DNA was extractedfrom each individual aphid using a DNA extraction kit (Qiagen, DNeasykit) according to manufacturer's instructions. DNA concentration wasmeasured using a nanodrop nucleic acid quantification, and Buchnera andaphid DNA copy numbers were measured by qPCR. The primers used forBuchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) andBuch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran,2016 PNAS). The primers used for aphid were ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

There was a Negative Response on Insect Fitness Upon Treatment with theScorpion AMPs

LSR-1 A. pisum 1^(st) and 2^(nd) instar aphids were divided into twoseparate treatment groups as defined in Experimental Design (above).Aphids were monitored daily and the number of aphids at eachdevelopmental stage was determined. Aphids treated with the negativecontrol alone began reaching maturity (5th instar stage) atapproximately 6 days (FIG. 38). Development was delayed in aphidstreated with Uy192, and after 8 days of treatment, aphids did not maturefurther than the 4^(rd) instar stage. These data indicate that treatmentwith Uy192 delayed and stopped progression of aphid development.

Treatment with Scorpion AMPs Results in Increased Aphid Mortality

Survival rate of aphids was also measured during the treatments. Themajority of the aphids treated with the control alone were alive at 3days post-treatment (FIG. 39). After 4 days, aphids began graduallydying, and some survived beyond 7 days post-treatment.

In contrast, aphids treated with Uy192 had lower survival rates thanaphids treated with control. These data indicate that there was adecrease in survival upon treatment with the scorpion AMP Ulyl92.

Treatment with Scorpion AMP Uy192 Results in Decreased Buchnera inAphids

To test whether treatment with Uy192, specifically resulted in loss ofBuchnera in aphids, and that this loss impacted aphid fitness, DNA wasextracted from aphids in each treatment group after 8 days of treatmentand qPCR was performed to determine the Buchnera/aphid copy numbers.Aphids treated with control alone had high ratios of Buchnera/aphid DNAcopies. In contrast, aphids treated with 100 ug/ml Uy192 in water had˜7-fold less Buchnera/aphid DNA copies (FIG. 40), indicating that Uy192treatment decreased Buchnera levels.

Together this data described previously demonstrated the ability to killand decrease the development, longevity, and endogenous bacterialpopulations, e.g., fitness, of aphids by treating them with a naturalscorpion antimicrobial peptide.

Example 21: Insects Treated with Scorpion Antimicrobial Peptides

This Example demonstrates the treatment of aphids with several scorpionantimicrobial peptides (AMPs) D10, D3, Uyct3, and Uy17, which have beenrecently identified AMPs in the venom gland transcriptome of thescorpion Urodacus yaschenkoi (Luna-Ramirez et al., 2017, Toxins). AMPstypically have a net positive charge and hence, a high affinity forbacterial membranes. This Example demonstrates that the effect of theAMPs on insects was mediated through the modulation of bacterialpopulations endogenous to the insect that were sensitive to AMPpeptides. One targeted bacterial strain is Buchnera aphidicola, anobligate symbiont of aphids.

Aphids are agricultural insect pests causing direct feeding damage tothe plant and serving as vectors of plant viruses. In addition, aphidhoneydew promotes the growth of sooty mold and attracts nuisance ants.The use of chemical treatments, unfortunately still widespread, leads tothe selection of resistant individuals whose eradication becomesincreasingly difficult.

Therapeutic Design:

The indicated peptide or peptide cocktail (see Aphid MicroinjectionExperimental Design and Leaf perfusion-Plant Experimental Designsections for details below) was directly microinjected into aphids ordelivered using a combination of leaf perfusion and delivery throughplants. As a negative control, aphids were microinjected with water (formicroinjection experiments) or placed on leaves injected with water andwater in tube (for leaf perfusion and plant delivery experiments). Thetreatment solutions consisted of 20 nl of 5 μg/μl of D3 or D10 dissolvedin water (for aphid microinjections) or 40 μg/ml of a cocktail of D10,Uy17, D3, and UyCt3 in water via leaf injection and through plant (inEppendorf tube).

Aphid Microinjection Experimental Design

Microinjection was performed using NanoJet III Auto-Nanoliter Injectorwith the in-house pulled borosilicate needle (Drummond Scientific;Cat#3-000-203-G/XL). Aphids (LSR-1 strain, Acyrthosiphon pisum) weregrown on fava bean plants (Vroma Vicia faba from Johnny's SelectedSeeds) in a climate-controlled incubator (16 h light/8 h darkphotoperiod; 60±5% RH; 25±2° C.). Prior to being used for aphid rearing,fava bean plants were grown in potting soil at 24° C. with 16 h of lightand 8 h of darkness. To limit maternal effects or health differencesbetween plants, 5-10 adults from different plants were distributed among10 two-week-old plants, and allowed to multiply to high density for 5-7days. Each treatment group had approximately the same number ofindividuals injected from each of the collection plants. Adult aphidswere microinjected into the ventral thorax with 20 nl of either water or100 ng (20 ul of a 5 ug/ml solution of peptide D3 or D10. Themicroinjection rate as 5 nl/sec. After injection, aphids were placed ina deep petri dish containing a fava bean leaf with stem in 2% agar.

Peptides were synthesized by Bio-Synthesis at >75% purity. 1 mg oflyophilized peptide was reconstituted in 500 μl of 80% acetonitrile, 20%water, and 0.1% TFA, 100 μl (100 μg) was aliquoted into 10 individualEppendorf tubes, and allowed to dry.

After 5 days of treatment, DNA was extracted from the remaining aphidsin each treatment group. Briefly, the aphid body surface was sterilizedby dipping the aphid into a 6% bleach solution for approximately 5seconds. Aphids were then rinsed in sterile water and DNA was extractedfrom each individual aphid using a DNA extraction kit (Qiagen, DNeasykit) according to manufacturer's instructions. DNA concentration wasmeasured using a nanodrop nucleic acid quantification, and Buchnera andaphid DNA copy numbers were measured by qPCR. The primers used forBuchnera were Buch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) andBuch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran,2016 PNAS). The primers used for aphid were ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

Microinjection of Aphids with Scorpion Peptides D3 and D10 Results inDecreased Insect Survival

LSR-1 A. pisum 1^(st) and 2^(nd) instar aphids were divided into threeseparate treatment groups as defined in Experimental Design (describedherein). Aphids were monitored daily and survival rate was determined.After 5 days of treatment, the aphids injected with the scorpionpeptides had lower survival rates compared to water injected controls(9, 35, and 45% survival for injection with D3, D10, and water,respectively) (FIG. 41). These data indicate that there was a decreasein survival upon treatment with the scorpion AMPs D3 and D10.

Microinjection of Aphids with Scorpion Peptides D3 and D10 Results in aReduction of Buchnera Endosymbionts

To test whether injection with the scorpion AMPs D3 and D10,specifically resulted in loss of Buchnera in aphids, and that this lossimpacted aphid fitness, DNA was extracted from aphids in each treatmentgroup 5 days after injection and qPCR was performed to determine theBuchnera/aphid copy numbers. Aphids injected with water alone had highratios of Buchnera/aphid DNA (47.4) while aphids injected with D3 andD10 had lower ratios of Buchnera/aphid DNA (25.3 and 30.9, respectively)(FIG. 42). These data indicate that treatment with scorpion peptides D3and D10 resulted in decreased levels of the bacterial symbiont Buchnera.

Leaf Perfusion-Plant Delivery Experimental Design:

eNASCO Aphids (which harbor Buchnera and Serratia), Acyrthosiphon pisumwere grown on fava bean plants (Vroma Vicia faba from Johnny's SelectedSeeds) as described above. For experiments, first and second instaraphids were collected from healthy plants and divided into 2 differenttreatment groups: 1) negative control (water treated), 2) The treatmentsolution consisted of 40 μg/ml of each D10, Uy17, D3, and UyCt3 inwater. Each treatment group received approximately the same number ofindividuals from each of the collection plants.

Peptides were synthesized, dissolved, and aliquoted, as described above.For treatment (see Therapeutic design), water was added to a 100 μgaliquot of peptide to obtain the desired final concentration (40 μg/ml).The four peptides were combined to make the peptide cocktail solution.This solution was used to perfuse into leaves via injection. Followinginjection, the stems of the injected leaves were placed into a 1.5 mlEppendorf tube which was then sealed with parafilm. This feeding systemwas then placed into a deep petri dish (Fisher Scientific, Cat#FB0875711) and aphids were applied to the leaves of the plant.

For each treatment, 41-49 aphids were placed onto each leaf. The feedingsystems were changed every 2-3 days throughout the experiment. Aphidswere monitored daily for survival and dead aphids were removed from thedeep petri dish when they were discovered.

Treatment with Cocktail of Scorpion Peptides Results in Increased AphidMortality

Survival rate of aphids was also measured during the treatments. After 6days of treatment, aphids treated with the peptide cocktail had lowersurvival rates compared to those treated with water, and after 9 daysthe effect is more evident (16 and 29% survival for peptide cocktail andwater treated, respectively) (FIG. 43). These data indicate that therewas a decrease in survival upon treatment with the cocktail of scorpionAMPs, and as shown in previous Examples these AMP decreased theendosymbiont levels in the aphids.

Together this data described previously demonstrated the ability to killand decrease the longevity and endogenous bacterial populations, e.g.,fitness, of aphids by treating them with single natural scorpionantimicrobial peptides or a peptide cocktail.

Example 22: Insects Treated with an Antimicrobial Peptide Fused to aCell Penetrating Peptide

This Example demonstrates the treatment of aphids with a fused scorpionantimicrobial peptide (AMP) (Uy192) to a cell penetrating peptidederived from a virus. The AMP Uy192 is one of several recentlyidentified AMPs in the venom gland transcriptome of the scorpionUrodacus yaschenkoi (Luna-Ramirez et al., 2017, Toxins). AMPs typicallyhave a net positive charge and hence, a high affinity for bacterialmembranes. To enhance the delivery of the scorpion peptide Uy192 intoaphid cells, the peptide was synthesized fused to a portion of thetransactivator of transcription (TAT) protein of human immunodeficiencyvirus I (HIV-1). Previous studies have shown that conjugating this cellpenetrating peptide (CPP) to other molecules increased their uptake intocells via transduction (Zhou et al., 2015 Journal of Insect Science andCermenati et al., 2011 Journal of Insect Physiology). This Exampledemonstrates that the effect of the fused peptide on insects wasmediated through the modulation of bacterial populations endogenous tothe insect that were sensitive to the antimicrobial peptide. Onetargeted bacterial strain is Buchnera.

Therapeutic Design

The scorpion peptide conjugated to the cell penetrating peptide andfluorescently tagged with 6FAM (Uy192+CPP+FAM) was formulated using acombination of leaf perfusion and delivery through plants. The controlsolution (water) or treatment solution (Uy192+CPP+FAM) was injected intothe leaf and placed in the Eppendorf tube. The treatment solutionincluded 100 μg/ml Uy192+CPP+FAM in water.

Leaf Perfusion-Plant Delivery Experimental Design

LSR-1 aphids, Acyrthosiphon pisum were grown on fava bean plants (VromaVicia faba from Johnny's Selected Seeds) in a climate-controlledincubator (16 h light/8 h dark photoperiod; 60±5% RH; 25±2° C.). Priorto being used for aphid rearing, fava bean plants were grown in pottingsoil at 24° C. with 16 h of light and 8 h of darkness. To limit maternaleffects or health differences between plants, 5-10 adults from differentplants were distributed among 10 two-week-old plants, and allowed tomultiply to high density for 5-7 days. For experiments, first instaraphids were collected from healthy plants and divided into 2 differenttreatment groups: 1) negative control (water treated), 2) Uy192+CPP+FAMtreated with 100 μg/ml Uy192+CPP+FAM in water. Each treatment groupreceived approximately the same number of individuals from each of thecollection plants.

For treatment (see Therapeutic design), Uy192+CPP+FAM (amino acidsequence: YGRKKRRQRRRFLSTIWNGIKGLL-FAM) was synthesized by Bio-Synthesisat >75% purity. 5 mg of lyophilized peptide was reconstituted in 1 ml of80% acetonitrile, 20% water, and 0.1% TFA, 50 μl (100 μg) was aliquotedinto individual Eppendorf tubes, and allowed to dry. For treatment (seeTherapeutic design), 1 ml of sterile water was added to a 100 μg aliquotof peptide to obtain the final concentration of Uy192+CPP+FAM (100μg/ml). The solution was then injected into the leaf of the plant andthe stem of the plant was placed into a 1.5 ml Eppendorf tube. Theopening of the tube was closed using parafilm. This feeding system wasthen placed into a deep petri dish (Fisher Scientific, Cat# FB0875711)and aphids were applied to the leaves of the plant. Epi fluorescenceimaging of the leaf confirmed that the leaves contained the greenfluorescently tagged peptide in their vascular system.

For each treatment, 30 aphids were placed onto each leaf in triplicate.The feeding systems were changed every 2-3 days throughout theexperiment. Aphids were monitored daily for survival and dead aphidswere removed from the deep petri dish when they were discovered. Inaddition, the developmental stage (1st, 2nd, 3rd, 4th, 5th, and 5R (5thinstar aphids that are reproducing) instar) was determined dailythroughout the experiment.

At 5 days post-treatment, DNA was extracted from several aphids in eachtreatment group. Briefly, the aphid body surface was sterilized bydipping the aphid into a 6% bleach solution for approximately 5 seconds.Aphids were then rinsed in sterile water and DNA was extracted from eachindividual aphid using a DNA extraction kit (Qiagen, DNeasy kit)according to manufacturer's instructions. DNA concentration was measuredusing a nanodrop nucleic acid quantification, and Buchnera and aphid DNAcopy numbers were measured by qPCR. The primers used for Buchnera wereBuch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) andBuch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran,2016 PNAS). The primers used for aphid were ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

Treatment with Scorpion Peptide Uy192 Fused to a Cell PenetratingPeptide Delayed and Stopped Progression of Aphid Development

LSR-1 A. pisum 1st instar aphids were divided into three separatetreatment groups as defined in Experimental Design (above). Aphids weremonitored daily and the number of aphids at each developmental stage wasdetermined. Development for both aphids treated with water and thosetreated with the scorpion peptide fused to the cell penetrating peptidewas similar for days 0 and 1 (FIG. 44). By day 2, however, controltreated aphids developed to either in the second or third instar stage,while some aphids treated with the scorpion peptide fused to the cellpenetrating peptide remained in the first instar stage (FIG. 44). Evenat 3 days post-treatment, some aphids treated with the scorpion peptidefused to the cell penetrating peptide remained in the first instar stage(FIG. 44). By 7 days post-treatment, the majority of the water treatedaphids developed to the 5th or 5th reproducing instar stage. Incontrast, only 50 percent of aphids treated with the scorpion peptidefused to the cell penetrating peptide developed to the 5th instar stage,while ˜42 and ˜8 percent of aphids remained at the 3rd or 4th instarstage, respectively (FIG. 44). These data indicate that treatment withthe scorpion peptide Uy192 fused to the cell penetrating peptide delayedand stopped progression of aphid development.

Treatment with the Scorpion Peptide Uy192 Fused to a Cell PenetratingPeptide Resulted in Increased Aphid Mortality

Survival rate of aphids was also measured during the treatments.Approximately 40% of aphids treated with the control alone survived the7-day experiment (FIG. 45). In contrast, survival was significantly lessfor aphids treated with 100 μg/ml Uy192+CPP+FAM (p=0.0036, by Log-RankMantel Cox test), with only 16% of aphids surviving to day 7 (FIG. 45).These data indicate that there was a decrease in survival upon treatmentwith the scorpion peptide Uy192 fused to a cell penetrating peptide.

Treatment with a Scorpion Peptide Fused to a Cell Penetrating PeptideResulted in Decreased Buchnera/Aphid DNA Ratios

To test whether treatment with treatment with Uy192+CPP+FAM,specifically resulted in loss of Buchnera in aphids, and that this lossimpacted aphid fitness, DNA was extracted from aphids in each groupafter 5 days of treatment, and qPCR was performed to determine theBuchnera/aphid copy numbers. Aphids treated with water had high ratios(˜134) of Buchnera/aphid DNA. In contrast, aphids treated with thescorpion peptide fused to the cell penetrating peptide had ˜1.8-foldless Buchnera/aphid DNA copies after 5 days of treatment (FIG. 46).These data indicate that treatment with the scorpion peptide fused to acell penetrating peptide decreased endosymbiont levels.

The Scorpion Peptide Fused to a Cell Penetrating Peptide Freely Enteredthe Bacteriocytes to Act Against Buchnera

To test whether the cell penetrating peptide aids in the delivery of thescorpion peptide into the bacteriocytes directly, isolated bacteriocyteswere directly exposed to the fusion protein and imaged. Thebacteriocytes were dissected from the aphids in Schneider's mediumsupplemented with 1% w/v BSA (Schneider-BSA medium), and placed in animaging well containing 20 ul of schneider's medium. A 100 uglyophilized aliquot of the scorpion peptide was resuspended in 100 ul ofthe schneider's medium to produce 1 mg/ml solution, and 5 ul of thissolution was mixed in to the well containing bacteriocytes. After 30 minof incubation at room temperature, the bacteriocytes were thoroughlywashed to eliminate any excess free peptide in the solution. Images ofthe bacteriocytes were captured before and after the incubation (FIG.47). The fusion peptide penetrated the bacteriocyte membranes and wasdirectly available to Buchnera.

Together, this data demonstrates the ability to kill and decrease thedevelopment, longevity, and endogenous bacterial populations, e.g.,fitness, of aphids by treating them with the scorpion antimicrobialpeptide Uy192 fused to a cell penetrating peptide.

Example 23: Insects Treated with Vitamin Analogs

This Example demonstrates the treatment of aphids with the provitaminpantothenol which is the alcohol analog of pantothenic acid (VitaminB5). Aphids have obligate endosymbiont bacteria, Buchnera, that helpthem make essential amino acids and vitamins, including Vitamin B5. Aprevious study has shown that pantothenol inhibits the growth ofPlasmodium falciparium by inhibition of the specific parasite kinases(Saliba et al., 2005). It was hypothesized that treating insects withpantothenol would be detrimental for the bacterial-insect symbiosistherefore affecting insect fitness. This Example demonstrates that thetreatment with pantothenol decreased insect fitness.

Therapeutic Design:

Pantothenol solutions were formulated depending on the delivery method:

1) In artificial diet through the plants: with 0 (negative control) or10 or 100 uM pantothenol formulated in an artificial diet (based on Akeyand Beck, 1971; see Experimental Design) without essential amino acids(2 mg/ml histidine, 2 mg/ml isoleucine, 2 mg/ml leucine, 2 mg/ml lysine,1 mg/ml methionine, 1.62 mg/ml phenylalanine, 2 mg/ml threonine, 1 mg/mltryptophan, and 2 mg/ml valine).

2) Leaf coating: 100 μl of 0.025% nonionic organosilicone surfactantsolvent Silwet L-77 in water (negative control) or 100 μl of 50 μg/ml ofrifampicin formulated in solvent solution was applied directly to theleaf surface and allowed to dry.

Plant Delivery Experimental Design

Aphids (eNASCO, Acyrthosiphon pisum) were grown on fava bean plants(Vroma Vicia faba from Johnny's Selected Seeds) in a climate-controlledincubator (16 h light/8 h dark photoperiod; 60±5% RH; 25±2° C.). Priorto being used for aphid rearing, fava bean plants were grown in pottingsoil at 24° C. with 16 h of light and 8 h of darkness. To limit maternaleffects or health differences between plants, 5-10 adults from differentplants were distributed among 10 two-week-old plants, and allowed tomultiply to high density for 5-7 days. For experiments, first and secondinstar aphids were collected from healthy plants and divided into 3different treatment groups: 1) artificial diet alone without essentialamino acids, 2) artificial diet alone without essential amino acids and10 uM pantothenol, and 3) artificial diet alone without essential aminoacids and 100 uM pantothenol. Each treatment group receivedapproximately the same number of individuals from each of the collectionplants.

The artificial diet used was made as previously published (Akey andBeck, 1971 Continuous Rearing of the Pea Aphid, Acyrthosiphon pisum, ona Holidic Diet), with and without the essential amino acids (2 mg/mlhistidine, 2 mg/ml isoleucine, 2 mg/ml leucine, 2 mg/ml lysine, 1 mg/mlmethionine, 1.62 mg/ml phenylalanine, 2 mg/ml threonine, 1 mg/mltryptophan, and 2 mg/ml valine), except neither diet included homoserineor beta-alanyltyrosine. The pH of the diets was adjusted to 7.5 with KOHand diets were filter sterilized through a 0.22 μm filter and stored at4° C. for short term (<7 days) or at −80° C. for long term.

Pantothenol (Sigma Cat#295787) solutions were made at 10 uM and 100 uMin artificial diet without essential amino acids, sterilized by passingthrough a 0.22 μm syringe filter, and stored at −20° C. For treatments(see Therapeutic design), the appropriate amount of stock solution wasadded to the artificial diet without essential amino acids to obtain afinal concentration of 10 or 100 uM pantothenol. The diet was thenplaced into a 1.5 ml Eppendorf tube with a fava bean stem with a leafand the opening of the tube was closed using parafilm. This artificialdiet feeding system was then placed into a deep petri dish (FisherScientific, Cat# FB0875711) and aphids were applied to the leaves of theplant.

For each treatment, 16-20 aphids were placed onto each leaf. Artificialdiet feeding systems were changed every 2-3 days throughout theexperiment. Aphids were monitored daily for survival and dead aphidswere removed from the deep petri dish housing the artificial feedingsystem when they were discovered.

In addition, the developmental stage (1st, 2nd, 3rd, 4th, 5th instar)was determined daily throughout the experiment. Once an aphid reachedthe 4th instar stage, they were given their own artificial feedingsystem in a deep petri dish so that fecundity could be monitored oncethey reached adulthood.

For adult aphids, new nymphs were counted daily and then discarded. Atthe end of the experiments, fecundity was determined as the mean numberof offspring produced daily once the aphid reached adulthood. Picturesof aphids were taken throughout the experiment to evaluate sizedifferences between treatment groups.

After 8 days of treatment, DNA was extracted from multiple aphids fromeach treatment group. Briefly, the aphid body surface was sterilized bydipping the aphid into a 6% bleach solution for approximately 5 seconds.Aphids were then rinsed in sterile water and DNA was extracted from eachindividual aphid using a DNA extraction kit (Qiagen, DNeasy kit)according to manufacturer's instructions. DNA concentration was measuredusing a nanodrop nucleic acid quantification, and Buchnera and aphid DNAcopy numbers were measured by qPCR. The primers used for Buchnera wereBuch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) andBuch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran,2016 PNAS). The primers used for aphid were ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

Vitamin Analog Treatment Delays Aphid Development

eNASCO 1st and 2nd instar aphids were divided into three separatetreatment groups as defined in Plant Delivery Experimental Design(described herein). Aphids were monitored daily and the number of aphidsat each developmental stage was determined. Aphids treated withartificial diet alone without essential amino acids began reachingmaturity (5th instar stage) at approximately 5 days (FIG. 48A).Development was delayed in aphids treated with pantothenol, especiallyat days two and three post-treatment (FIG. 48A), indicating thattreatment with pantothenol impairs aphid development. Eventually, mostaphids from each treatment group reached maturity and began reproducing.In addition to monitoring developmental stage of aphids over time,aphids were also imaged and aphid area was determined. All aphids werethe same size after 1 day of treatment, however, by 3 dayspost-treatment, aphids treated with pantothenol were smaller in areathan untreated controls. Moreover, aphids treated with pantothenol hadmuch less of an increase in body size (as determined by area) over thecourse of the experiment, compared to aphids treated with artificialdiet alone without essential amino acids (FIG. 48B).

Vitamin Analog Treatment Increased Aphid Mortality

Survival rate of aphids was also measured during the treatments. Aphidsreared on artificial diet alone without essential amino acids had highersurvival rates compared to aphids treated with 10 or 100 uM pantothenol(FIG. 49), indicating that pantothenol treatment negatively affectedaphid fitness.

Treatment with Pantothenol Decreases Aphid Fecundity

Fecundity was also monitored in aphids during the treatments. Thefraction of aphids surviving to maturity and reproducing was determined.Approximately one quarter of aphids treated with artificial diet withoutessential amino acids survived to reach maturity by 8 dayspost-treatment (FIG. 50A). In contrast, only a little over 1/10th ofaphids treated with 10 or 100 uM pantothenol survived to reach maturityand reproduce by 8 days post-treatment. The mean day aphids in eachtreatment group began reproducing was also measured and for alltreatment groups, the mean day aphids began reproducing was 7 days (FIG.50B). Additionally, the mean number of offspring per day produced bymature, reproducing aphids was also monitored. Aphids treated withartificial diet alone without essential amino acids producedapproximately 7 offspring/day. In contrast, aphids treated with 10 and100 uM pantothenol only produced approximately 4 and 5 offspring/day,respectively, shown in FIG. 50C. Taken together, these data indicatethat pantothenol treatment resulted in a loss of aphid reproduction.

Pantothenol Treatment does not Affect Buchnera in Aphids

To test whether treatment with pantothenol, specifically resulted inloss of Buchnera in aphids, and that this loss impacted aphid fitness,DNA was extracted from aphids in each treatment group after 8 days oftreatment and qPCR was performed to determine the Buchnera/aphid copynumbers. Aphids treated with artificial diet alone without essentialamino acids had high ratios of Buchnera/aphid DNA copies as did aphidstreated with each of the two concentrations of pantothenol (FIG. 51).These data indicate that pantothenol treatment does not affectBuchneralaphid DNA copy number directly.

Leaf Coating Delivery Experimental Design:

Pantothenol powder was added to 0.025% of a nonionic organosiliconesurfactant solvent, Silwet

L-77, to obtain a final concentration of 10 uM pantothenol. Thetreatment was filter sterilized using a 0.22 um filter and stored at 4degrees C. Aphids (eNASCO strain, Acyrthosiphon pisum) were grown onfava bean plants as described in a previous Example. For experiments,first instar aphids were collected from healthy plants and divided into2 different treatment groups: 1) negative control (solvent solutiononly) and 2) 10 uM pantothenol in solvent. 100 ul of the solution wasabsorbed onto a 2×2 cm piece of fava bean leaf.

Each treatment group received approximately the same number ofindividuals from each of the collection plant. For each treatment, 20aphids were placed onto each leaf. Aphids were monitored daily forsurvival and dead aphids were removed when they were discovered. Inaddition, the developmental stage (1st, 2nd, 3rd, 4th, 5th instar, and5R, representing a reproducing 5th instar) was determined dailythroughout the experiment.

Pantothenol treatment delivered through leaf coating does not affectaphid development

eNASCO 1st instar aphids were divided into two separate treatment groupsas defined in the Experimental Design described herein. Aphids weremonitored daily and the number of aphids at each developmental stage wasdetermined. Aphids placed on coated leaves treated with either thecontrol or pantothenol solution matured at similar rates up to two dayspost-treatment (FIG. 52). These data indicate that leaf coating withpantothenol did not affect aphid development.

Pantothenol Treatment Delivered Through Leaf Coating Increased AphidMortality

Survival rate of aphids was also measured during the leaf coatingtreatments. Aphids placed on coated leaves with pantothenol had lowersurvival rates than aphids placed on coated leaves with the controlsolution (FIG. 53). These data indicate that pantothenol treatmentdelivered through leaf coating significantly (p=0.0019) affected aphidsurvival. All aphids died in this experiment and there were no remainingaphids left to extract DNA from and determine Buchnera/aphid DNA ratios.

Together this data described in the previous Examples demonstrate theability to kill and decrease the development, reproductive ability,longevity, and endogenous bacterial populations, e.g., fitness, ofaphids by treating them with pantothenol through multiple deliverymethods.

Example 24: Insects Treated with a Cocktail of Amino Acid TransportersInhibitors

This Example demonstrates the treatment of aphids with a cocktail ofamino acid analogs. The objective of this treatment was to inhibituptakes of glutamine into the bacteriocytes through the ApGLNT1glutamine transporter. It has previously been shown that arginineinhibits glutamine uptake by the glutamine transporter (Price et al.,2014 PNAS), and we hypothesized that treatment with analogs of arginine,or other amino acid analogs, may also inhibit uptake of essential aminoacids into the aphid bacteriocytes. This Example demonstrates that thedecrease in fitness upon treatment was mediated through the modulationof bacterial populations endogenous to the insect that were sensitive toamino acid analogs. One targeted bacterial strain is Buchnera.

Therapeutic Design:

The amino acid cocktail was formulated for delivery through leafperfusion and through the plant. This delivery method consisted ofinjecting leaves with approximately 1 ml of the amino acid cocktail inwater (see below for list of components in the cocktail) or 1 ml of thenegative control solution containing water only.

Leaf Perfusion and Delivery Through Plants Experimental Design:

Aphids LSR-1 (which harbor only Buchnera), Acyrthosiphon pisum weregrown on fava bean plants (Vroma Vicia faba from Johnny's SelectedSeeds) in a climate-controlled incubator (16 h light/8 h darkphotoperiod; 60±5% RH; 25±2° C.). Prior to being used for aphid rearing,fava bean plants were grown in potting soil at 24° C. with 16 h of lightand 8 h of darkness. To limit maternal effects or health differencesbetween plants, 5-10 adults from different plants were distributed among10 two-week-old plants, and allowed to multiply to high density for 5-7days. For experiments, first instar aphids were collected from healthyplants and divided into 2 different treatment groups: 1) negativecontrol (water treatment) and 2) amino acid cocktail treatment. Theamino acid cocktail contained each of the following agents at theindicated final concentrations: 330 μM L-NNA (N-nitro-L-Arginine;Sigma), 0.1 mg/ml L-canavanine (Sigma), 0.5 mg/ml D-arginine (Sigma),0.5 mg/ml D-phenylalanine (Sigma), 0.5 mg/ml D-histidine (Sigma), 0.5mg/ml D-tryptophan (Sigma), 0.5 mg/ml D-threonine (Sigma), 0.5 mg/mlD-valine (Sigma), 0.5 mg/ml D-methionine (Sigma), 0.5 mg/ml D-leucine,and 6 μM L-NMMA (citrate) (Cayman Chemical). ˜1 ml of the treatmentsolution was perfused into the fava bean leaf via injection and the stemof the plant was put into a 1.5 ml Eppendorf tube containing thetreatment solution. The opening of the tube was closed using parafilm.This feeding system was then placed into a deep petri dish (FisherScientific, Cat# FB0875711) and aphids were applied to the leaves of theplant. For each treatment, a total of 56-58 aphids were placed onto eachleaf (each treatment consisted of two replicates of 28-31 aphids). Eachtreatment group received approximately the same number of individualsfrom each of the collection plants. The feeding systems were changedevery 2-3 days throughout the experiment. Aphids were monitored dailyfor survival and dead aphids were removed from the deep petri dish whenthey were discovered. The aphid developmental stage (1st, 2nd, 3rd, 4th,and 5th instar) was determined daily throughout the experiment andmicroscopic images were taken of the aphids on day 5 to determine aphidarea measurements.

Stock solutions of L-NNA were made at 5 mM in water, sterilized bypassing through a 0.22 μm syringe filter, and stored at −20° C. Stocksolutions of L-canavanine were made at 100 mg/ml in water, sterilized bypassing through a 0.22 μm syringe filter, and stored at 4° C. Stocksolutions of D-arginine and D-threonine were made at 50 mg/ml in water,sterilized by passing through a 0.22 μm syringe filter, and stored at 4°C. Stock solutions of D-valine and D-methionine were made at 25 mg/ml inwater, sterilized by passing through a 0.22 μm syringe filter, andstored at 4° C. Stock solutions of D-leucine were made at 12 mg/ml inwater, sterilized by passing through a 0.22 μm syringe filter, andstored at 4° C. Stock solutions of D-phenylalanine and D-histidine weremade at 50 mg/ml in 1M HCl, sterilized by passing through a 0.22 μmsyringe filter, and stored at 4° C. Stock solutions of D-tryptophan weremade at 50 mg/ml in 0.5M HCl, sterilized by passing through a 0.22 μmsyringe filter, and stored at 4° C. Stock solutions of L-NMMA were madeat 6 mg/ml in sterile PBS, sterilized by passing through a 0.22 μmsyringe filter, and stored at −20° C. For treatments (see Therapeuticdesign), the appropriate amount of stock solution was added to water toobtain the final concentration of the agent in the cocktail as indicatedabove.

After 6 days of treatment, DNA was extracted from multiple aphids fromeach treatment group. Briefly, the aphid body surface was sterilized bydipping the aphid into a 6% bleach solution for approximately 5 seconds.Aphids were then rinsed in sterile water and DNA was extracted from eachindividual aphid using a DNA extraction kit (Qiagen, DNeasy kit)according to manufacturer's instructions. DNA concentration was measuredusing a nanodrop nucleic acid quantification, and Buchnera and aphid DNAcopy numbers were measured by qPCR. The primers used for Buchnera wereBuch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) andBuch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran,2016 PNAS). The primers used for aphid were ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

Treatment with a Cocktail of Amino Acid Analogs Delayed and StoppedProgression of Aphid Development

LSR-1 1st instar aphids were divided into two separate treatment groupsas defined in Leaf perfusion and delivery through plants experimentaldesign (described herein). Aphids were monitored daily and the number ofaphids at each developmental stage was determined. Aphids treated withwater began reaching maturity (5th instar stage) at day 5 post-treatment(FIG. 54A). By 6 days post-treatment, ˜20 percent of aphids treated withwater reached the 5th instar stage. In contrast, less than 3 percent ofthe aphids treated with the amino acid cocktail reached the 5th instarstage, even after 6 days (FIG. 54A). This delay in development upontreatment with the amino acid cocktail was further exemplified by aphidsize measurements taken at 5 days post-treatment. Aphids treated withwater alone were approximately 0.45 mm2, whereas aphids treated with theamino acid cocktail were approximately 0.33 mm2 (FIG. 54B). These dataindicate that treatment with the amino acid cocktail delayed aphiddevelopment, negatively impacting aphid fitness.

Treatment with an Amino Acid Analog Cocktail Resulted in DecreasedBuchnera in Aphids

To test whether treatment with the amino acid analog cocktailspecifically resulted in loss of Buchnera in aphids, and that this lossimpacted aphid fitness, DNA was extracted from aphids in each treatmentgroup after 6 days of treatment and qPCR was performed to determine theBuchnera/aphid copy numbers. Aphids placed on control solution had highratios of Buchnera/aphid DNA copies. In contrast, aphids placed on AAcocktail treatment had a drastic reduction of Buchnera/aphid DNA copies(FIG. 55), indicating that the AA analog cocktail treatment eliminatedendosymbiotic Buchnera.

Together, this data demonstrates the ability to decrease the developmentand endogenous bacterial populations, e.g., fitness, of aphids bytreating them with a cocktail of amino acid analogs.

Example 25: Insects Treated with a Combination of Agents (Antibiotic,Peptide, and Natural Antimicrobial)

This Example demonstrates the treatment of insects with a combination ofthree antimicrobial agents—an antibiotic (rifampicin), a peptide (thescorpion peptide Uy192), and a natural antimicrobial (low molecularweight chitosan). In other Examples, each of these agents administeredindividually resulted in decreased aphid fitness and reducedendosymbiont levels. This Example demonstrates that through the deliveryof a combination of treatments, insect fitness and endosymbiont levelswere reduced as well as, or better than, treatment with each individualagent alone.

Therapeutic Design

The combination treatment was formulated for delivery through leafperfusion and through the plant. This delivery method consisted ofinjecting leaves with approximately 1 ml of the combination treatment inwater (with final concentrations of 100 μg/ml rifampicin, 100 μg/mlUy192, and 300 μg/ml chitosan) or 1 ml of the negative control solutioncontaining water only.

Leaf Perfusion and Delivery Through Plants Experimental Design

Aphids LSR-1 (which harbor only Buchnera), Acyrthosiphon pisum weregrown on fava bean plants (Vroma Vicia faba from Johnny's SelectedSeeds) in a climate-controlled incubator (16 h light/8 h darkphotoperiod; 60±5% RH; 25±2° C.). Prior to being used for aphid rearing,fava bean plants were grown in potting soil at 24° C. with 16 h of lightand 8 h of darkness. To limit maternal effects or health differencesbetween plants, 5-10 adults from different plants were distributed among10 two-week-old plants, and allowed to multiply to high density for 5-7days. For experiments, first instar aphids were collected from healthyplants and divided into 2 different treatment groups: 1) negativecontrol (water treatment) and 2) a combination of 100 μg/ml rifampicin,100 μg/ml Uy192, and 300 μg/ml chitosan treatment. ˜1 ml of thetreatment solution was perfused into the fava bean leaf via injectionand the stem of the plant was put into a 1.5 ml Eppendorf tubecontaining the treatment solution. The opening of the tube was closedusing parafilm. This treatment system was then placed into a deep petridish (Fisher Scientific, Cat# FB0875711) and aphids were applied to theleaves of the plant. For each treatment, a total of 56 aphids wereplaced onto each leaf (each treatment consisted of two replicates of 28aphids). Each treatment group received approximately the same number ofindividuals from each of the collection plants. The feeding systems werechanged every 2-3 days throughout the experiment. Aphids were monitoreddaily for survival and dead aphids were removed from the deep petri dishwhen they were discovered. The aphid developmental stage (1^(st)2^(nd),3^(rd), 4^(th), and 5^(th) instar) was determined daily throughout theexperiment and microscopic images were taken of the aphids on day 5 todetermine aphid area measurements.

Rifampicin (Tokyo Chemical Industry, LTD) stock solution was made at 25mg/ml in methanol, sterilized by passing through a 0.22 μm syringefilter, and stored at −20° C. For treatment, the appropriate amount ofstock solution was added to water to obtain a final concentration of 100μg/ml rifampicin. Uy192 was synthesized by Bio-Synthesis at >75% purity.1 mg of lyophilized peptide was reconstituted in 500 μl of 80%acetonitrile, 20% water, and 0.1% TFA. 100 μl (100 μg) was aliquotedinto 10 individual Eppendorf tubes and allowed to dry. For treatment, 1ml of water was added to a 100 μg aliquot of peptide to obtain the finalconcentration of 100 μg/ml Uy192. Chitosan (Sigma, catalog number448869-50G) stock solution was made at 1% in acetic acid, sterilizedautoclaving, and stored at 4° C. For treatments the appropriate amountof stock solution was added to water to obtain the final concentrationof 300 μg/ml chitosan.

After 6 days of treatment, DNA was extracted from multiple aphids fromeach treatment group. Briefly, the aphid body surface was sterilized bydipping the aphid into a 6% bleach solution for approximately 5 seconds.Aphids were then rinsed in sterile water and DNA was extracted from eachindividual aphid using a DNA extraction kit (Qiagen, DNeasy kit)according to manufacturer's instructions. DNA concentration was measuredusing a nanodrop nucleic acid quantification, and Buchnera and aphid DNAcopy numbers were measured by qPCR. The primers used for Buchnera wereBuch_groES_18F (CATGATCGTGTGCTTGTTAAG; SEQ ID NO: 238) andBuch_groES_98R (CTGTTCCTCGAGTCGATTTCC; SEQ ID NO: 239) (Chong and Moran,2016 PNAS). The primers used for aphid were ApEF1a 107F(CTGATTGTGCCGTGCTTATTG; SEQ ID NO: 240) and ApEF1a 246R(TATGGTGGTTCAGTAGAGTCC; SEQ ID NO: 241) (Chong and Moran, 2016 PNAS).qPCR was performed using a qPCR amplification ramp of 1.6 degrees C./sand the following conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 55° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

Treatment with a Combination of Three Antimicrobial Agents Delayed andStopped Progression of Aphid Development

LSR-1 1^(st) instar aphids were divided into two separate treatmentgroups as defined in Leaf perfusion and delivery through plantsexperimental design (described herein). Aphids were monitored daily andthe number of aphids at each developmental stage was determined. Aphidstreated with water began reaching maturity (5th instar stage) at day 5post-treatment (FIG. 56A). By 6 days post-treatment, ˜20 percent ofaphids treated with water reached the 5th instar stage. In contrast, noaphids treated with the combination of three agents reached the 5thinstar stage, even after 6 days (FIG. 56A). This delay in developmentupon combination treatment was further exemplified by aphid sizemeasurements taken at 5 days post-treatment. Aphids treated with wateralone were approximately 0.45 mm², whereas aphids treated with the3-agent combination were approximately 0.26 mm² (FIG. 56B). These dataindicate that treatment with a combination of agents delayed aphiddevelopment, negatively impacting aphid fitness.

Treatment with a Combination of Three Antimicrobial Agents IncreasedAphid Mortality

Survival was also monitored daily after treatment. At 2 dayspost-treatment, approximately 75 percent of aphids treated with waterwere alive, whereas only 62 percent of aphids treated with thecombination of agents were alive. This trend of more aphids survivingtreatment in the control (water-treated) group continued for theduration of the experiment. At 6 days post-treatment, 64 percent ofcontrol (water-treated) aphids survived, whereas 58 percent of aphidstreated with a combination of rifampicin, Uy192, and chitosan survived(FIG. 57). These data indicate that the combination of treatmentsnegatively affected aphid survival.

Treatment with a Combination of Three Agents Resulted in DecreasedBuchnera in Aphids

To test whether treatment with a combination of a peptide, antibiotic,and natural antimicrobial specifically resulted in loss of Buchnera inaphids, and that this loss impacted aphid fitness, DNA was extractedfrom aphids in each treatment group after 6 days of treatment and qPCRwas performed to determine the Buchnera/aphid copy numbers. Aphidstreated with water alone ratios of approximately 2.3 Buchnera/aphid DNA(FIG. 58). In contrast, aphids treated with the combination of apeptide, antibiotic, and natural antimicrobial had approximately 2-foldlower ratios of Buchnera/aphid DNA (FIG. 58). These data indicate thatcombination treatment reduced endosymbiont levels, which resulted indecreased aphid fitness.

Together, this data demonstrates the ability to decrease the developmentand endogenous bacterial populations, e.g., fitness, of aphids bytreating them with a combination of a peptide, antibiotic, and naturalantimicrobial.

Example 26: Insects Treated with an Antibiotic Solution

This Example demonstrates the effects of treatment of weevils withciprofloxacin, a bactericidal antibiotic that inhibits the activity ofDNA gyrase and topoisomerase, two enzymes essential for DNA replication.This Example demonstrates that the phenotypic effect of ciprofloxacin onanother model insect, weevils, was mediated through the modulation ofbacterial populations endogenous to the insects that were sensitive tociprofloxacin. One targeted bacterial strain is Sitophilus primaryendosymbiont (SPE, Candidatus Sodalis pierantonius).

Experimental Design:

Sitophilus maize weevils (Sitophilus zeamais) were reared on organiccorn at 27.5° C. and 70% relative humidity. Prior to being used forweevil rearing, corn was frozen for 7 days and then tempered to 10%humidity with sterile water. For experiments, adult male/female matingpairs were divided into 3 different treatment groups that were done intriplicate: 1) water control, 2) 250 μg/ml ciprofloxacin, and 3) 2.5mg/ml ciprofloxacin. Ciprofloxacin (Sigma) stock solutions were made at25 mg/ml in 0.1 N HCl, sterilized by passing through a 0.22 μm syringefilter, and stored at −20° C. For treatments, the appropriate amount ofstock solution was diluted in sterile water.

The weevils were subjected to three successive treatments:

-   -   1. The first treatment included soaking 25 g of corn with each        of the three treatment groups listed above: 1) water control, 2)        250 μg/ml ciprofloxacin, and 3) 2.5 mg/ml ciprofloxacin.        Briefly, 25 g of corn was placed into a 50 ml conical tube and        each of the treatment was added to fill the tube completely. The        tube was put on a shaker for 1.5 hours after which, the corn was        removed and placed into a deep petri dish and air dried.        Male/Female mating pairs were then added to each treatment group        and allowed to feed for 4 days.    -   2. After 4 days, mating pairs were removed and subjected to a        second treatment by putting them onto 25 g of new corn treated        with 1) water control, 2) 250 μg/ml ciprofloxacin, and    -   3) 2.5 mg/ml ciprofloxacin. Mating pairs fed and laid eggs on        this corn for 7 days. The corn from the second treatment was        assessed for the emergence of offspring (see assessment of        offspring, below)    -   3. Mating pairs were subjected to a final treatment which        included a combination of submerging them into the treatment (1)        water control, 2) 250 μg/ml ciprofloxacin, and 3) 2.5 mg/ml        ciprofloxacin for 5 seconds and then placing them in a vial with        10 corn kernels that had been coated with 1 ml of 1) water        control, 2) 250 μg/ml ciprofloxacin, and 3) 2.5 mg/ml        ciprofloxacin.

Weevil survival was monitored daily for 18 days, after which DNA wasextracted from the remaining weevils in each group. Briefly, the weevilbody was surface sterilized by dipping the weevil into a 6% bleachsolution for approximately 5 seconds. Weevils were then rinsed insterile water and DNA was extracted from each individual aphid using aDNA extraction kit (Qiagen, DNeasy kit) according to manufacturer'sinstructions. DNA concentration was measured using a nanodrop nucleicacid quantification, and SPE and weevil DNA copy numbers were measuredby qPCR. The primers used for SPE were qPCR_Sod_F (ATAGCTGTCCAGACGCTTCG;SEQ ID NO: 242) and qPCR_Sod_R (ATGTCGTCGAGGCGATTACC; SEQ ID NO: 243).The primers used for weevil (β-actin) were SACT144_FOR(GGTGTTGGCGTACAAGTCCT; SEQ ID NO: 244) and SACT314_REV(GAATTGCCTGATGGACAGGT; SEQ ID NO: 245) (Login et al., 2011). qPCR wasperformed using a qPCR amplification ramp of 1.6 degrees C./s and thefollowing conditions: 1) 95° C. for 10 minutes, 2) 95° C. for 15seconds, 3) 57° C. for 30 seconds, 4) repeat steps 2-3 40×, 5) 95° C.for 15 seconds, 6) 55° C. for 1 minute, 7) ramp change to 0.15 degreesC./s, 8) 95° C. for 1 second. qPCR data was analyzed using analytic(Thermo Fisher Scientific, QuantStudio Design and Analysis) software.

Assessment of Offspring:

After 25 days, one replicate of the corn kernels from the secondtreatment of the adult mating pairs was dissected (see ExperimentalDesign, above) to check for the presence of any developing larvae,pupae, or adult weevils. Most of the development of Sitophilus weevilstakes place within the grain/rice/corn and adults emerge from thekernels once their development is complete. Corn kernels were gentlydissected open with a scalpel and any developing weevils were collectedand the percent of adults, pupae, and larvae were determined. Theweevils from the dissection were then surface sterilized and the levelsof SPE were determined by qPCR. Corn kernels from the remaining tworeplicates of each of the groups from the second treatment were notdissected but checked daily for the emergence of adult weevils.

Assessment of Antibiotic Penetration into Corn

25 mg of corn kernels was placed into a 50 ml conical tube and water or2.5 mg/ml or 0.25 mg/ml ciprofloxacin in water was added to fill thetube. The kernels were soaked for 1.5 hours as described herein. Aftersoaking, kernels were air dried and assayed to determine whether theantibiotic was able to coat and penetrate the kernel. To test this, aconcentrated sample of Escherichia coli DH5a in water was spread onto 5Luria Broth (LB) plates. To each plate the following was done, 1) a cornkernel soaked in water was added, 2) an entire corn kernel that had beensoaked with 2.5 or 0.25 mg/ml ciprofloxacin was added, and 3) a half ofcorn kernel that had been soaked with 2.5 or 0.25 mg/ml ciprofloxacinwas added and placed inside down on the plate. The plates were incubatedovernight at 37 degrees C. and bacterial growth and/or zone(s) ofinhibition were assessed the next day.

Soaking Corn Kernels in Antibiotics Allowed Antibiotics to Coat theSurface and Penetrate Corn Kernels.

To test whether ciprofloxacin could coat the surface of a corn kernelafter a kernel, corn kernels were soaked in water without antibiotics orwater with 2.5 or 0.25 mg/ml ciprofloxacin (as described above). Aconcentrated culture of E. coli was then spread onto LB plates and oneof the coated kernels was then placed onto the center of the plate. Theplates were incubated overnight, and bacterial growth was assessed thenext day.

A lawn of bacteria grew on the entire plate with the corn kernel thathad been coated in water without any antibiotics (FIG. 56A). Incontrast, no bacteria grew on plates with entire corn kernels that hadbeen soaked in either of the two concentrations of ciprofloxacin (FIG.56B, left panels). These data show that the coating method employed inthese experiments allowed for ciprofloxacin to successfully coat thesurface of corn kernels and inhibit bacterial growth.

To test whether ciprofloxacin could penetrate the corn kernel, cornkernels soaked in 2.5 or 0.25 mg/ml ciprofloxacin were cut in half andplaced cut side down on an LB plate with a concentrated culture of E.coli. The plates were incubated overnight and the next day bacterialgrowth was assessed. No bacterial growth was present on the plates withthe kernels soaked in either concentration of antibiotic, indicatingthat ciprofloxacin penetrated the corn kernel (FIG. 56B, right panels).Together, these data indicate that the method of corn kernel soakingused for these experiments successfully coated and penetrated thekernels with the antibiotic.

Antibiotic Treatment Decreases SPE Levels in the F0 Generation.

S. zeamais mating pairs were divided into three separate treatmentgroups as defined in Experimental Design (above). Weevils were monitoreddaily and all weevils remained alive for the course of the experiment.After 18 days of treatment, weevils were surface sterilized, genomic DNAwas extracted, and SPE levels were measured by qPCR. Weevils treatedwith water only had approximately 4 and 8-fold higher amounts of SPEcompared to weevils treated with 250 ug/ml and 2.5 mg/ml ciprofloxacin,respectively (FIG. 57). These data indicate that treatment of weevilswith ciprofloxacin resulted in decreased levels of SPE.

Antibiotic Treatment Delays the Development and Decreases the SPE Levelsof the F1 Generation of Weevils.

The development of the F1 generation of weevils was assessed bydissecting corn kernels that F0 mating pairs had oviposited on for 7days and were subsequently removed. After 25 days, 12 offspring werefound in water/control-treated corn with the majority (˜67%) ofoffspring being in the pupae form (FIG. 58A). 13 and 20 offspring werefound in weevils treated with 250 ug/ml and 2.5 mg/ml ciprofloxacin,respectively. Interestingly, weevils treated with antibiotic showed adelay in development compared to control treated weevils with themajority (38 and 65% for 250 ug/ml and 2.5 mg/ml ciprofloxacin,respectively) of the offspring being in the larval form (FIG. 58A).

Genomic DNA was extracted from weevils dissected from the corn kernelsand qPCR was performed to measure the levels of SPE. Water treated F1weevils had approximately 4-fold higher levels of SPE compared toweevils treated with 2.5 mg/ml ciprofloxacin (FIG. 58B). These dataindicate that treatment with ciprofloxacin reduced the levels of the SPEin weevils which led to a delay in development.

Antibiotic Treatment Decreased Weevil Reproduction

The number of weevils that emerged over the course of 43 days after theinitial mating pairs were removed from the second treatment was used ameasure for the fecundity FIGS. 59A and 59B). The first weevil emergedon day 29, and the total number of weevils that emerged till day 43 werecounted. While weevils treated with water and 250 ug/ml had similaramount of F1 offspring, there were much less offspring that emerged fromthe 2.5 mg/ml treatment group, indicating that antibiotic treatmentdecreased SPE levels affected weevil fecundity.

Together with the previous Examples, this data demonstrate the abilityto kill and decrease the development, reproductive ability, longevity,and endogenous bacterial populations, e.g., fitness, of weevils bytreating them with an antibiotic through multiple delivery methods.

Example 27: Mites Treated with an Antibiotic Solution

This Example demonstrates the ability to kill, decrease the fitness oftwo-spotted spider mites by treating them with rifampicin, a narrowspectrum antibiotic that inhibits DNA-dependent RNA synthesis byinhibiting a bacterial RNA polymerase, and doxycycline, a broad-spectrumantibiotic that prevents bacterial reproduction by inhibiting proteinsynthesis. The effect of rifampicin and doxycycline on mites wasmediated through the modulation of bacterial populations endogenous tothe mites that were sensitive to the antibiotics.

Insects, such as mosquitoes, and arachnids, such as ticks, can functionas vectors for pathogens causing severe diseases in humans and animalssuch as Lyme disease, dengue, trypanosomiases, and malaria. Vector-bornediseases cause millions of human deaths every year. Also, vector-bornediseases that infect animals, such as livestock, represent a majorglobal public health burden. Thus, there is a need for methods andcompositions to control insects and arachnids that carry vector-bornediseases. Two-spotted spider mites are arachnids in the same subclass asticks. Therefore, this Example demonstrates methods and compositionsused to decrease the fitness of two-spotted spider mites and provideinsight into decreasing tick fitness.

Therapeutic Design

Two treatments were used for these experiments 1) 0.025% Silwet L-77(negative control) or 2) a cocktail of antibiotics containing 250 μg/mlrifampicin and 500 μg/ml doxycycline. Rifampicin (Tokyo ChemicalIndustry, LTD) stock solutions were made at 25 mg/ml in methanol,sterilized by passing through a 0.22 μm syringe filter, and stored at−20° C. Doxcycline (manufacturer) stock solutions were made at 50 mg/mLin water, sterilized by passing through a 0.22 μm syringe filter, andstored at −20° C.

Experimental Design:

This assay tested an antibiotic solution on two-spotted spider mites anddetermined how their fitness was altered by targeting endogenousmicrobes.

Kidney plants were grown in potting soil at 24° C. with 16 h of lightand 8 h of darkness. Mites were reared on kidney bean plants at 26° C.and 15-20% relative humidity. For treatments, one-inch diameter leafdisks were cut from kidney bean leaves and sprayed with either 0.025%Silwet L-77 (negative control) or the antibiotic cocktail (250 μg/mlrifampicin and 500 μg/ml doxycycline in 0.025% Silwet L-77) using aMaster Airbrush Brand Compressor Model C-16-B Black Mini Airbrush AirCompressor. The compressor was cleaned with ethanol before, after, andbetween treatments. The liquid was feed through the compressor using aquarter inch tube. A new tube was used for each treatment.

After leaf discs dried, four of each treatment were placed in a cup ontop of a wet cotton ball covered with a piece of kimwipe. Each treatmentsetup was done in duplicate. 25 adult female mites were then placed inthe cup. On day 4, the females were removed from the cup and the eggsand larvae were left on the leaf discs.

On day 11, mites at the protonymph stage and the deutonymph stage weretaken from the cups and placed in their own tube so survival could bemeasured. Each tube contained a moist cotton ball covered with a pieceof kimwipe with a half inch leaf disc treated with the negative controlor the cocktail.

The mites were observed under a dissecting microscope daily afterfeeding on a leaf treated with the antibiotic or the control solutions,and classified according to the following categories:

-   -   Alive: they walked around when on their legs or moved after        being poked by a paint brush.    -   Dead: immobile and did not react to stimulation from a paint        brush

A sterile paint brush was used to stimulate the mites by touching theirlegs. Mites classified as dead were kept throughout the assay andrechecked for movement daily. The assays were carried out at 26° C. and15-20% relative humidity.

Antibiotic Treatment Increased Mite Mortality

The survival rates of the two-spotted spider mites treated with theantibiotic cocktail were compared to the mites treated with the negativecontrol. The survival rates of the mites treated with the cocktail weredecreased compared to the control (FIG. 60).

This data demonstrates the ability to decrease fitness of mites bytreating them with a solution of antibiotics.

Example 28: Insects Treated with a Solution of Purified Phage

This Example demonstrates the isolation and purification of phages fromenvironmental samples that targeted specific insect bacteria. ThisExample also demonstrates the efficacy of isolated phages against thetarget bacteria in vitro by plaque assays, by measuring their oxygenconsumption rate, and the extracellular acidification rate. Finally,this Example demonstrates the efficacy of the phages in vivo, bymeasuring the ability of the phage to the target bacteria from flies bytreating them with a phage isolated against the bacteria. This Exampledemonstrates that a pathogenic bacterium that decreased the fitness ofan insect can be cleared using a phage to target the bacteria.Specifically, Serratia marcescens which is a pathogenic bacterium inflies can be cleared with the use of a phage that was isolated fromgarden compost.

Experimental Design

Isolation of Specific Bacteriophages from Natural Samples:

Bacteriophages against target bacteria were isolated from environmentalsource material. Briefly, a saturated culture of Serratia marcescens wasdiluted into fresh double-strength tryptic soy broth (TSB) and grown for˜120 minutes to early log-phase at 24-26° C., or into double-strengthLuria-Bertani (LB) broth and grown for ˜90 min at 37° C. Garden compostwas prepared by homogenization in PBS and sterilized by 0.2 μmfiltration. Raw sewage was sterilized by 0.2 μm filtration. One volumeof filtered source material was added to log-phase bacterial culturesand incubation was continued for 24 h. Enriched source material wasprepared by pelleting cultures and filtering supernatant fluid through0.45 μm membranes.

Phages were isolated by plating samples onto double-agar bacteriallawns. Stationary bacterial cultures were combined with molten 0.6% agarLB or TSB and poured onto 1.5% agar LB or TSB plates.

After solidification, 2.5 μL of phage sample dilutions were spotted ontothe double-agar plates and allowed to absorb. Plates were then wrappedand incubated overnight at 25° C. (TSA) or 37° C. (LB), then assessedfor the formation of visible plaques. Newly isolated plaques werepurified by serial passaging of individual plaques on the target strainby picking plaques into SM Buffer (50 mM Tris-HCl [pH 7.4], 10 mM MgSO4,100 mM NaCl) and incubating for 15 min at 55° C., then repeating thedouble-agar spotting method from above using the plaque suspension.

Bacteriophages were successfully isolated from both sewage and compost,as detailed above. Plaque formation was clearly evident after spottingsamples onto lawns of the S. marcescens bacteria used for theenrichments.

Passaging, Quantification, and Propagation of Bacteriophages:

Propagation and generation of phage lysates for use in subsequentexperiments was performed using bacteriophages isolated and purified asabove. Briefly, saturated bacterial cultures were diluted 100-fold intofresh medium and grown for 60-120 minutes to achieve anearly-logarithmic growth state for effective phage infection. Phagesuspensions or lysates were added to early log phase cultures andincubation was continued until broth clearing, indicative of phagepropagation and bacterial lysis, was observed, or until up to 24 hpost-infection. Lysates were harvested by pelleting cells at 7,197×g for20 min, then filtering the supernatant fluid through 0.45 or 0.2 μmmembranes. Filtered lysates were stored at 4° C.

Enumeration of infective phage particles was performed using thedouble-agar spotting method. Briefly, a 1:10 dilution series of sampleswas performed in PBS and dilutions were spotted onto solidifieddouble-agar plates prepared with the host bacteria as above.Plaque-forming units (PFU) were counted after overnight incubation todetermine the approximate titer of samples.

In Vitro Analysis of Isolated Phages Measuring Bacterial Respiration:

A Seahorse XFe96 Analyzer (Agilent) was used to measure the effects ofphages on bacteria by monitoring oxygen consumption rate (OCR) andextracellular acidification rate (ECAR) during infection. XFe96 plateswere coated the day prior to experiments by 15 μL of a 1 mg/mLpoly-L-lysine stock per well and dried overnight at 28° C. and XFe96probes were equilibrated by placing into wells containing 200 μL of XFCalibrant and incubating in the dark at room temperature. The followingday, poly-L-lysine coated plates were washed twice with ddH2O. Saturatedovernight cultures of E. coli BL21 (LB, 37° C.) or S. marcescens (TSB,25° C.) were subcultured at 1:100 into the same media and grown withaeration for ˜2.5 h at 30° C. Cultures were then diluted to O.D.600nm˜0.02 using the same media. Treatments were prepared by dilutingstocks into SM Buffer at 10× final concentration and loading 20 μL ofthe 10× solutions into the appropriate injection ports of the probeplate. While the probes were equilibrating in the XFe96 Flux Analyzer,bacterial plates were prepared by adding 90 μL of bacterial suspensionsor media controls and spun at 3,000 rpm for 10 min. Followingcentrifugation, an additional 90 μL of the appropriate media were addedgently to the wells so as not to disturb bacterial adherence, bringingthe total volume to 180 μL per well.

The XFe96 Flux Analyzer was run at ˜30° C., following a Mix, Wait, Readcycling of 1:00, 0:30, 3:00. Four cycles were completed to permitequilibration/normalization of bacteria, then the 20 μL treatments wereinjected and cycling continued as above, for a total time ofapproximately 6 h. Data were analyzed using the Seahorse XFe96 Wavesoftware package.

The effects of isolated bacteriophages were assayed by measuring oxygenconsumption rate (OCR) and extracellular acidification rate (ECAR) ofbacteria with a Seahorse XFe96 Analyzer. When E. coli was infected withphage T7 and S. marcescens infected with the newly isolated ϕSmVL-C1,dramatic decreases in OCR were observed following brief bursts in thisrate (FIG. 64). For both phages with both host organisms, the Seahorseassay permitted the detection of successful phage infection without theneed for plaque assays. Thus, this method is applicable for detectingphage infection of a host organism not amenable to traditional phagedetection methods.

SYBR Gold Transduction Assay for Infection Identification:

Bacteriophage preparations were prepared for staining by pretreatingwith nucleases to remove extraviral nucleic acids that could interferewith fluorescent signal interpretation. Briefly, MgCl2 was added to 10mL of phage lysate at 10 mM final concentration, and RNase A (Qiagen)and DNase I (Sigma) were both added to final concentrations of 10 μg/mL.Samples were incubated for 1 h at room temperature. After nucleasetreatment, 5 mL of lysates were combined with 1 μL of SYBR Gold (Thermo,10,000×) and incubated at room temperature for ˜1.5 h. Excess dye wassubsequently removed from samples using Amicon ultrafiltration columns.Briefly, Amicon columns (15 mL, 10 k MWCO) were washed by adding 10 mLof SM Buffer and spinning at 5,000×g, 4° C. for 5 min. Labeled phagesamples were then spun through the columns at 5,000×g, 4° C. until thevolume had decreased by approximately 10-fold (15-30 min). To washsamples, 5 mL SM Buffer was added to each reservoir and the spinrepeated, followed by two additional washes. After the third wash, theretained samples were pipetted out from the Amicon reservoirs andbrought up to approximately 1 mL using SM Buffer. To remove largercontaminants, washed and labeled phage samples were spun at 10,000×g for2 min, and the supernatants were subsequently filtered through 0.2 μmmembranes into black microtubes and stored at 4° C.

Saturated bacterial cultures (E. coli MG1655 grown in LB at 37° C., S.marcescens and S. symbiotica grown in TSB at 26° C.) were prepared byspinning down 1 mL aliquots and washing once with 1 mL PBS before afinal resuspension using 1 mL PBS. Positive control labeled bacteriawere stained by combining 500 μL of washed bacteria with 1 μL of SYBRGold and incubating for 1 h in the dark at room temperature. Bacteriawere pelleted by spinning at 8,000×g for 5 min and washed twice with anequal volume of PBS, followed by resuspension in a final volume of 500μL PBS. A volume of 25 μL of stained bacteria was combined with 25 μL ofSM Buffer in a black microtube, to which 50 μL of 10% formalin (5% finalvolume, ˜2% formaldehyde) was added and mixed by flicking. Samples werefixed at room temperature for ˜3 h and then washed using Amiconultrafiltration columns. Briefly, 500 μL of picopure water was added toAmicon columns (0.5 mL, 100 k MWCO) and spun at 14,000×g for 5 min towash membranes. Fixed samples were diluted by adding 400 μL of PBS andthen transferred to pre-washed spin columns and spun at 14,000×g for 10min. Columns were transferred to fresh collection tubes, and 500 μL ofPBS was added to dilute out fixative remaining in the retentate.Subsequently, two additional PBS dilutions were performed, for a totalof three washes. The final retentates were diluted to roughly 100 μL,then columns were inverted into fresh collection tubes and spun at1,000×g for 2 min to collect samples. Washed samples were transferred toblack microtubes and stored at 4° C.

For transduction experiments and controls, 25 μL of bacteria (or PBS)and 25 μL of SYBR Gold labeled phage (or SM Buffer) were combined inblack microtubes and incubated static for 15-20 min at room temperatureto permit phage adsorption and injection into recipient bacteria.Immediately after incubation, 50 μL of 10% formalin was added to samplesand fixation was performed at room temperature for ˜4 h. Samples werewashed with PBS using Amicon columns, as above.

Injection of bacteriophage nucleic acid was required for a phage tosuccessfully infect a host bacterial cell. Coliphage P1kc labeled withSYBR Gold and co-incubated with S. marcescens revealed the presence offluorescent bacteria by microscopy, validating the use of this assay ina phage isolation pipeline. As with the Seahorse assay, this approachprovided an alternative to traditional phage methods to permit expansionto organisms not amenable to plaque assay. Additionally, the SYBR Goldtransduction assay did not require bacterial growth, so is applicable toanalysis of phages targeting difficult or even non-culturable organisms,including endosymbionts such as Buchnera.

Testing In Vivo Efficacy of the Phages Against S. marcescens inDrosophila melanogaster Flies

S. marcescens cultures were grown in Tryptic Soy Broth (TSB) at 30° C.with constant shaking at 200 rpm.

The media used to rear fly stocks was cornmeal, molasses and yeastmedium (11 g/l yeast, 54 g/l yellow cornmeal, 5 g/l agar, 66 ml/lmolasses, and 4.8 ml/l propionic acid). All the components of the dietexcept propionic acid were heated together to 80° C. in deionized waterwith constant mixing for 30 minutes and let to cool to 60° C. Propionicacid was then mixed in and 50 ml of the diet was aliquoted intoindividual bottles and allowed to cool down and solidify. The flies wereraised at 26° C., 16:8 hour light:dark cycle, at around 60% humidity.

To infect the flies with S. marcescens, a fine needle (About 10 um widetip) was dipped in a dense overnight stationary phase culture and thethorax of the flies was punctured. For this experiment, four replicatesof 10 males and 10 females each were infected with S. marcescens usingthe needle puncturing method as the positive control for fly mortality.For the treatment group, four replicates of 10 males and 10 females eachwere pricked with S. marcescens and a phage solution containing about108 phage particles/ml. Finally, two replicates of 10 males and 10females each that were not pricked or treated in anyway were used as anegative control for mortality.

Flies in all conditions were placed in food bottles and incubated at 26°C., 16:8 light:dark cycle, at 60% humidity. The number of alive and deadflies were counted every day for four days after the pricking. All Theflies pricked with S. marcescens alone were all dead within 24 hours ofthe treatment. In comparison, more than 60% of the flies in the phagetreatment group, and all the flies in the untreated control group werealive at that time point (FIG. 65). Further, most of the flies in thephage treatment group and the negative control group went on to survivefor four more days when the experiment was terminated.

To ascertain the reason of death of the flies, dead flies from both theS. marcescens and S. marcescens+phage pricked flies were homogenized andplated out. Four dead flies from each of the four replicates of both theS. marcescens and the S. marcescens+phage treatment were homogenized in100 ul of TSB. A 1:100 dilution was also produced by diluting thehomogenate in TSB. 10 ul of the concentrated homogenate as well as the1:100 dilution was plated out onto TSA plates, and incubated overnightat 30° C. Upon inspection of the plates for bacteria growth, all theplates from the dead S. marcescens pricked flies had a lawn of bacteriagrowing on them, whereas the plates from the dead S. marcescens+phagepricked flies had no bacteria on them. This shows that in the absence ofthe phage, S. marcescens likely induced septic shock in the fliesleading to their fatality. However, in the presence of the phage, themortality may have been due to injury caused by the pricking with theneedle.

Other Embodiments

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. A method of decreasing fitness of a vector for a human pathogen, themethod comprising: delivering an antimicrobial peptide having at least90% sequence identity with one or more of the following: cecropin (SEQID NO: 82), melittin, copsin, drosomycin (SEQ ID NO: 93), dermcidin (SEQID NO: 81), andropin (SEQ ID NO: 83), moricin (SEQ ID NO: 84),ceratotoxin (SEQ ID NO: 85), abaecin (SEQ ID NO: 86), apidaecin (SEQ IDNO: 87), prophenin (SEQ ID NO: 88), indolicidin (SEQ ID NO: 89),protegrin (SEQ ID NO: 90), tachyplesin (SEQ ID NO: 91), or defensin (SEQID NO: 92) to the vector.
 2. The method of claim 1, wherein the deliverycomprises delivering the antimicrobial peptide to at least one habitatwhere the vector grows, lives, reproduces, feeds, or infests.
 3. Themethod of claim 1, wherein the antimicrobial peptide is delivered in aninsect comestible composition for ingestion by the vector.
 4. The methodof claim 1, wherein the antimicrobial peptide is formulated as a liquid,a solid, an aerosol, a paste, a gel, or a gas composition.
 5. The methodof claim 1, wherein the insect is at least one of a mosquito, midge,louse, sandfly, tick, triatomine bug, tsetse fly, or flea.
 6. Acomposition comprising an antimicrobial peptide having at least 90%sequence identity with one or more of the following: cecropin, melittin,copsin, drosomycin, dermcidin, andropin, moricin, ceratotoxin, abaecin,apidaecin, prophenin, indolicidin, protegrin, tachyplesin, or defensinformulated for targeting a microorganism in a vector for a humanpathogen.
 7. The composition of claim 6, wherein the antimicrobialpeptide is at a concentration of about 0.1 ng/g to about 100 mg/g in thecomposition.
 8. The composition of claim 6, wherein the antimicrobialpeptide further comprises a targeting domain.
 9. The composition ofclaim 6, wherein the antimicrobial peptide further comprises a cellpenetrating peptide.